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Biomedical subjects

D Neumann-Haefelin

Publications and source records attributed to D Neumann-Haefelin.

At least 37 records · Page 2Linked to original sources
PubMed1995

Phylogenetic analysis of primate foamy viruses by comparison of pol sequences.

The relationship between primate foamy viruses was determined by comparing a 425-bp DNA segment obtained by PCR using primers homologous to highly conserved portions of the pol gene. The phylogenetic tree of 14 foamy viruses investigated reflects the relationship between their host species: A cluster of Asian Old World monkey foamy viruses including simian foamy virus (SFV) prototypes 1 and 2 (isolated from Macaca cyclopsis) is separated from African Old World foamy viruses including prototype SFV-3 and SFV-3 strain LK-3 (isolated from African green monkeys, Cercopithecus aethiops). These two clusters of Old World monkey foamy viruses are more distantly related to a cluster of ape and human foamy viruses including prototypes SFV-6, SFV-7, SFV cpz (all isolated from chimpanzees), and human foamy virus (HFV). The New World prototype SFV-8 (isolated from a spider monkey, Ateles sp.) is distinct from the Old World cluster. Our own foamy virus isolates from a rhesus monkey (Macaca mulatta) and an African green monkey were grouped to the Asian or the African Old World monkey foamy virus cluster, respectively. The foamy virus sequences obtained from lymphocytes of two humans, one exposed to African green monkeys and the other to cultured HFV, were compared. The first sequence was closely related to the African Old World monkey foamy virus cluster, whereas the second was identical to HFV, except for a single mismatch. We conclude that limited sequencing of amplified DNA is a powerful tool for classification as well as molecular epidemiology of foamy viruses.

Animals↗
PubMed1995

Laboratory diagnosis of HCMV-related disease in renal transplant patients - pp65 antigen detection versus nested PCR.

BACKGROUND: Sixty-five renal transplant (Tx) recipients were monitored for signs and symptoms of human cytomegalovirus (HCMV) infection. OBJECTIVES: Different diagnostic markers were evaluated for early and correct diagnosis of HCMV disease. STUDY DESIGN: Blood and urine samples were obtained in weekly intervals and the following markers were determined: (1) IgG and IgM antibodies in serum using immunofluorescence and ELISA tests; (2) viral shedding in urine by rapid centrifugation culture (RCC); (3) viral antigen (pp65) in peripheral blood leukocytes (PBL) by immunofluorescence and (4) viral DNA in PBL by nested PCR (NPCR). RESULTS: Twenty-two patients remained free of HCMV infection, 18 patients developed clinical symptoms of HCMV disease, and 25 patients remained asymptomatic in spite of laboratory signs of HCMV infection. For the early detection of HCMV disease, the highest sensitivity was achieved using NPCR (100%) and pp65 antigen detection (94%). RCC and IgM serology were less sensitive (62% and 40% respectively). The differences of sensitivity were significant. Clinical specificity was 47% for NPCR, 79% for pp65 antigen detection, 66% for RCC, and 68% for IgM serology. CONCLUSION: In contrast to NPCR, pp65 antigen detection was closely correlated with the appearance of clinical disease and proved to be a useful marker in the monitoring of antiviral therapy.

Journal Article↗
PubMed1995

Markers of foamy virus infections in monkeys, apes, and accidentally infected humans: appropriate testing fails to confirm suspected foamy virus prevalence in humans.

Foamy viruses (FVs) persist in healthy individuals of various mammalian species, including nonhuman primates. Laboratory markers of FV infection are (1) virus in throat epithelium or peripheral blood lymphocytes (PBLs), (2) proviral DNA sequences in PBLs and various solid organs, and (3) antibodies reactive to viral antigens on Western blots, in radioimmunoprecipitation tests, and in immunofluorescence assays. Using PCR and serological tests, we readily detected FV markers in naturally infected African green monkeys, rhesus monkeys, and chimpanzees, as well as in accidentally infected humans. Transmission of simian foamy viruses to humans (by bite or inadvertent laboratory infection) leads to viral markers, without affecting the recipient. Reports on FV-associated clinical disorders (e.g., thyroid or neurological) have remained controversial. In this study we failed to detect, by PCR, viral sequences in the samples from 223 patients, including 16 HIV-infected Africans, 46 Graves' disease patients, and 28 patients with the de Quervain's thyroiditis. Evaluation of 2688 sera from suspected high-risk areas (e.g., Central and East Africa, or high-risk groups such as HIV-infected individuals and patients with AIDS, thyroid, and neurological disorders) did not reveal FV-specific antibodies in a single case. Previously reported FV seroprevalence in various populations has never been verified by appropriate confirmatory tests. The strain of "human foamy virus" has remained a unique isolate. In conclusion, FVs are unlikely--at present--to circulate in human populations.

Africa↗
PubMed1995

Human cytomegalovirus immediate early and late transcripts in peripheral blood leukocytes: diagnostic value in renal transplant recipients.

Peripheral blood leukocytes of renal transplant recipients were investigated to compare the following markers of human cytomegalovirus (HCMV) infection: pp65 antigen by indirect immunofluorescence, viral DNA by nested polymerase chain reaction (PCR), and immediate early (IE) and late (pp150) mRNA by nested PCR following reverse transcription. Sixty-five patients were monitored weekly for 20 weeks after transplantation. In 76 samples from 20 patients positive for HCMV DNA by PCR, HCMV mRNA was detected. Detectable amounts of IE and pp150 mRNA were positively correlated with high numbers of pp65 antigen-positive cells and confirmed the significance of pp65 antigen as a marker for active viral replication. However, with respect to the early diagnosis of HCMV-related disease and monitoring of antiviral therapy, the test for viral mRNA was not superior to the pp65 antigen test.

Base Sequence↗
PubMed1995

Human endogenous retrovirus K10: expression of Gag protein and detection of antibodies in patients with seminomas.

The human endogenous retrovirus K10 (HERV-K10) has been identified in the human genome by its homology to retroviruses of other vertebrates (M. Ono, T. Yasunaga, T. Miyata, and H. Ushikubo, J. Virol. 60:589-598, 1986). Using PCR amplification, DNA cloning, sequencing, and procaryotic expression, we were able to demonstrate that HERV-K10 encodes a 73-kDa protein which was processed by a HERV-K10-encoded protease to yield proteins p22/p26, p30, and p15/16. Analysis of the teratocarcinoma cell line Tera 1 or tumor tissues by immunoblotting demonstrated that the 80-kDa polyprotein of HERV-K10 gag and a processed protein of 39 kDa were expressed. In addition, a major protein of 39 kDa and additional species of 30, 22, 19, and 17 kDa could be detected in the supernatant of Tera 1 cells, suggesting that HERV-K10 Gag proteins are either secreted or processed to probably incomplete viral particles. In addition, the gag gene of HERV-K10 was expressed in the baculovirus system. Using this recombinant system to test antisera from patients with different diseases and healthy individuals, we were able to detect antibodies against the N-terminal part of HERV-K10 Gag in 2 to 4% of groups of tumor patients with titers ranging between 1:80 and 1:640, while approximately 0.1 to 0.5% of healthy individuals exhibited antibodies with lower titers. In contrast, patients with seminoma had antibody titers in the range of 1:2,560 at the time when the tumor was detected. Immunohistochemistry using specific rabbit sera or monoclonal antibodies against HERV-K10 Gag revealed that the Gag protein is expressed in the cytoplasm of the tumor cells. Furthermore, an 80-kDa protein corresponding to the HERV-K10 Gag polyprotein could be detected in tumor biopsies. For the first time, these data indicate that HERV-K10 Gag proteins are synthesized in seminoma cells and tumors exhibit relatively high antibody titers against Gag. So far, no information on which role HERV-K10 plays in the development of this tumor exists.

Antibodies, Viral↗
PubMed1995

Liver failure due to disseminated HSV-1 infection in a newborn twin.

Most cases of neonatal herpes simplex virus (HSV) infection result from contact with maternal genital tract secretions and are caused by infections with HSV type 2. We report on a fatal HSV-1 infection in a newborn twin presenting with liver failure. The infection was acquired by single contact with an aunt. The route of transmission was proven by PCR followed by restriction endonuclease fingerprinting and DNA sequencing. This report demonstrates that liver failure may be an early and single symptom in life-threatening neonatal HSV-1 infection.

DNA, Viral↗
PubMed1995

Detection of DNA in single cells using an automated cell deposition unit and PCR.

A highly effective single-cell PCR method using a fluorescence-activated cell sorting (FACS)-based automated cell deposition unit (ACDU) that sorts single cells directly into PCR tubes was developed. To evaluate the sensitivity of this method, single ACH-2 cells (containing one HIV-1 genome per cell) were sorted, and 220 out of 228 samples (96.5%) were HIV DNA-positive by PCR. Furthermore, the number of samples accidentally containing more than one cell was determined by sorting single cells from a mixture of human cytomegalovirus (HCMV)-infected fibroblasts and ACH-2 cells. Multiplex nested PCR (nPCR) was then performed, detecting HCMV and HIV DNA simultaneously. From 66 sorted cells, 2 (3%) were double-positive for HIV and HCMV, 31 (47%) for HCMV alone, 30 (45.5%) for HIV alone and 3 (4.5%) were PCR-negative. The ACDU was then programmed to sort defined numbers of cells into PCR tubes. This is similar to classic dilution assays in that it allows the determination of the percentage of cells that was positive for a specific DNA. The accuracy of multiple cell deposition by the ACDU was evaluated by determining the percentage of HIV-positive cells in defined mixtures of ACH-2 and uninfected H9 cells. Infection rates determined by the ACDU correlated well with the rates expected from the given dilutions.

Autoanalysis↗
PubMed1994

Antiviral activity of African medicinal plants.

Plants from Africa and Mauritius with a history of use in traditional medicine have been investigated for their antiviral activities. Extracts were tested against poliovirus, herpes simplex virus and rhinovirus in plaque reduction assays. Their general toxicity and effects on interferon production were also studied.

Animals↗
PubMed1994

Absence of foamy virus DNA in Graves' disease.

A report on the high prevalence of foamy virus DNA in lymphocytes from French patients with Graves' disease prompted us to investigate a similar cohort of 41 German patients. Using PCR amplification and Southern blot hybridization, we detected foamy virus DNA only in lymphocytes of two accidentally infected humans and five naturally infected monkeys, as well as in DNA samples from four Graves' disease patients investigated in the French study. However, we failed to detect foamy virus DNA in peripheral blood lymphocytes from any of the 41 Graves' disease patients of the German cohort. Thus, a causative role of foamy viruses in this thyroid disease is highly improbable.

Adult↗
PubMed1994

Reactivity of primate sera to foamy virus Gag and Bet proteins.

In order to establish criteria for the serodiagnosis of foamy virus infections we investigated the extent to which sera from infected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays. Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein. Both the Gag precursor molecules of 70 and 74K apparent M(r) and the cytoplasmic 60K M(r) Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells. Rabbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey and African green monkey origin. This was reflected by a broad cross-reactivity of the respective monkey sera to the Gag proteins of the various foamy virus isolates. Cross-reactivity of antisera against the Bet protein was restricted to viruses from man and the great apes. Recombinant Gag and Bet proteins expressed in prokaryotes or in insect cells were readily recognized by foamy virus-positive primate sera. Screening serum samples from chimpanzees with HFV Gag and Bet proteins expressed by recombinant baculoviruses revealed that 18 out of 35 (52%) were positive for Gag antibodies. Of these, 13 (72%) showed antibodies against the Bet protein, indicating that Bet antigen is of value in serological screening for foamy virus infections.

Animals↗
PubMed1993

Regulatory elements in the long terminal repeat (LTR) of simian foamy virus type 3 (SFV-3).

Simian foamy virus type 3 (SFV-3) is a retrovirus that has a complex genome organization and encodes two open reading frames (ORF-1 and ORF-2) in addition to the genes coding for gag, pol, and env. In this report, we demonstrate that ORF-1 of SFV-3 encodes a transcriptional transactivator designated taf (transactivator of foamy virus) which augments gene expression directed by the viral long terminal repeat (LTR). The taf responsive elements have been mapped to the U3 region of the LTR, between positions -637 and -180 (+1 represents the transcription initiation site). Two regions between -637 and -180 in the LTR are targets for taf transactivation. These target sequences for taf confer responsiveness to a heterologous promoter independent of orientation; thus, they function like conditional enhancers. The R-U5 region of the viral LTR is shown to have an inhibitory effect on gene expression. SFV-1 is a related spumavirus and encodes a taf gene that augments expression directed by the SFV-3 LTR as well as the SFV-1 LTR; however, the taf gene of SFV-3 transactivates the SFV-3 LTR but not the SFV-1 LTR. These data on regulatory elements in the SFV-3 LTR show that the mechanism of foamy virus transactivation is significantly different from lentiviruses as well as from the HTLV group of viruses.

Cell Line↗
PubMed1993

Simian foamy virus type 3 (SFV-3) in latently infected Vero cells: reactivation by demethylation of proviral DNA.

Cell cultures latently infected with simian foamy virus type 3 (SFV-3) were established by suppressing lytic infection in Vero cells with 3'-azido-3'-deoxythymidine (AZT) and homologous antibodies (African green monkey serum immune to SFV-3). The resulting cell line, designated Vero-L, was shown to contain at least one copy per cell of SFV-3 DNA stably integrated at a defined site of the host cell genome. Sequencing of 669 bp at the integration site did not identify a coding region and revealed a 4-bp imperfect repeat in host cell DNA due to SFV-3 integration. Over 2 years of subcultivation, no spontaneous expression of proviral genes could be detected. However, the demethylating agent 5'-azacytidine reactivated lytic infection, proving conservation of the complete viral genome. Comparison of proviral DNA from latently and lytically infected cells supports the notion that methylation is instrumental in keeping SFV-3 infection in latency.

Animals↗
PubMed1993

Foamy viruses.

Foamy viruses share complex genome organization with lentiviruses and certain oncoviruses. The open reading frame 3' of env encodes a transcriptional transactivator. Distinct responsive sequences were identified in the long terminal repeats (LTRs) of simian (SFV-1 and SFV-3) and human foamy viruses (HFV). Transactivation of heterologous LTRs was described including those of simian and human immunodeficiency viruses. Foamy viruses persist for the whole lifetime in infected hosts (primates, cats, hamsters, cattle, and probably other mammals). The virus may be orally shed and transmitted, while being latent in various internal organs. Selective viral gene expression in the brains of mice transgenic for HFV has suggested a particular relationship to neural tissue. In latently SFV-3-infected cultured cells, methylation of proviral DNA is apparently involved in the control of latency. Demethylation as well as transfection with the transactivator were shown to be instrumental in viral reactivation. Natural infections with foamy viruses are common, elicit strong immune responses, and seem to be asymptomatic in nonhuman primates. Detection of such infections, however, may not be a triviality in man. While accidental transmission of foamy viruses to man is well documented, reported seroprevalence in human populations and the association of HFV with specific pathology (e.g. thyroiditis de Quervain, amyotrophic lateral sclerosis, and Graves' disease) are controversial and remain to be proven.

Animals↗
PubMed1992

Genomic organization and expression of simian foamy virus type 3 (SFV-3).

The complete nucleotide sequence of simian foamy virus type 3 (SFV-3) strain LK-3, isolated from an African green monkey, was determined. In addition to translation frames representing the gag, pol, and env genes, two open reading frames are located in the region between the env gene and the 3' long terminal repeat (LTR). Both SFV-3 and SFV-1 encode two open reading frames between env and the 3' LTR, whereas HFV encodes three open reading frames in this region. Northern blot analysis of cell cultures infected with SFV-3 revealed subgenomic RNAs for these open reading frames. The protease of SFV-3 is encoded by the pol gene in contrast to HFV which encodes the protease in the gag gene. Notably, the pol gene of SFV-3 in the +1 translational frame relative to the gag gene; this observation is in agreement with SFV-1, but differs for HFV and all other retrovirus genomes reported. Thus, gag-pol precursors of the SFVs appear to be expressed by a +1 frameshift. Nucleotide and deduced amino acid alignments of SFV-3, SFV-1, and HFV revealed an unexpected homology pattern; highest homologies are observed in the pol and env genes but low homologies are noted in the gag genes and the additional open reading frames. Analysis of phylogenetic trees confirms the classification of foamy viruses as a subfamily of retroviruses, distinct from the lentiviruses and oncoviruses.

Amino Acid Sequence↗
PubMed1992

[Neurologic complications of Q-fever (Coxiella burnetii)].

We report on fifteen patients with a typical syndrome of Q-fever. Four patients (= 27%) showed signs of nervous system involvement. One patient developed a meningitic syndrome, another patient had a passing psychosis. In two patients, severe cerebellar deficits were predominant. It is pointed out that these two patients were under lithium therapy because of manic-depressive illness. A synergetic neurotoxic effect of cociella burneti infection and lithium is discussed as a possible cause of the severe cerebellar pattern of symptoms.

Adult↗
PubMed1992

Diagnosis and management of the acute retinal necrosis syndrome.

The acute retinal necrosis (ARN) syndrome is an increasingly occurring entity characterized by the triad of acute confluent peripheral retinitis with papillitis and anterior-chamber uveitis. We present case reports on four patients (age, 12-65 years) with an ARN syndrome caused by herpes simplex or varicella zoster virus and discuss diagnostic and therapeutic modalities. Immediate antiviral therapy in three patients exhibiting the typical clinical features reduced the intraocular inflammation. However, due to proliferative vitreoretinopathy with peripheral retinal necrosis, vitrectomy with encircling band and silicone oil instillation was necessary in all patients. The suspected diagnosis of an ARN syndrome induced by herpes simplex virus (HSV) was confirmed in one case during the early stage of the disease by the detection of increased levels of HSV-IgA in the vitreous and in another case by the measurement of increased titers of HSV-IgG in the vitreous. For the first time, we found intraocular HSV DNA sequences using the polymerase chain reaction (PCR) in one of these patients. In a fourth patient intraocular varicella zoster virus (VZV) infection was confirmed by the detection of elevated VZV-IgA levels and by positive PCR in the intraocular fluids. Two patients who were diagnosed and treated early retained a visual acuity of 0.4 and 0.5, respectively, whereas in the other two patients, whose diagnosis and therapy were delayed (> 6 weeks), visual acuity was reduced to light perception. We conclude that use of the PCR in the intraocular fluids together with detection of autochthonous antibodies in the vitreous seem to be the most important diagnostic laboratory tools.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1991

Colonization of adrenal glands and ovaries of mice by variants of HSV 1 and 2. II. Histopathological, immunohistochemical and in situ hybridization studies.

The herpes simplex virus (HSV)-infected mouse model was used to correlate histopathological lesions in adrenal glands and ovaries with the localisation of viral nucleic acids and viral antigens, employing in situ hybridization and immunohistochemistry. In the adrenals, the lesions were mainly restricted to the zona fasciculata and the zona reticularis, sometimes extending to the medulla. In the ovaries, lesions were detected in follicles and in the stroma. During the course of infection, HSV nucleic acids could be detected earlier than HSV proteins. Next to the center of necrotic foci mainly HSV proteins were detected, whereas peripheral cells were found to contain viral nucleic acids. In situ hybridization revealed no proof of HSV latency in either organ. Among HSV-1 and HSV-2 strains of different neurovirulence, only HSV-2 variant ER- failed to replicate in adrenal glands and ovaries, whereas the neuroinvasive variant ER+ showed the same patterns as the HSV-1 strains used.

Adrenal Glands↗