ABSTRACT Betaretroviruses exist in endogenous and exogenous forms in hosts that are widely distributed and evolutionarily distantly related. Here we report the discovery and characterization of several previously unknown betaretrovirus groups in the genomes of Mus musculus and Rattus norvegicus . Each group contains both mouse and rat elements, and several of the groups are more closely related to previously known betaretroviruses from nonmurine hosts. Some of the groups also include members from hosts which were not previously known to harbor betaretroviruses, such as the gray mouse lemur ( Microcebus murinus ) and Seba's short-tailed bat ( Carollia perspicillata ). Some of the mouse and rat elements possess intact open reading frames for gag , pro , pol , and/or env genes and display characteristics of having retrotransposed recently. We propose a model whereby betaretroviruses have been evolving within the genomes of murid rodents for at least the last 20 million years and, subsequent to (or concomitant with) the global spread of their murid hosts, have occasionally been transmitted to other species.

For diagnosing invasive aspergillosis (IA), an increasing clinical problem in immunocompromised patients, molecular tools are gaining in importance. Detection of Aspergillus DNA in blood samples was investigated by a nested PCR assay in a murine model of experimentally induced IA. Ex vivo, the detection threshold of the PCR assay was determined in blood and organ homogenates of mice. After intravenous injection of Aspergillus fumigatus conidia on different days, growth of colonies was determined in cultures of blood and organs from immunocompetent and immunosuppressed mice and Aspergillus DNA was detected from blood samples by a nested PCR assay. The detection threshold of the PCR assay was as low as 1 c.f.u. ml(-1). The assay proved to be more sensitive than cultures of blood, with sensitivity rates between 17(.)6 and 87(.)5 % depending on the fungal burden. In conclusion, the nested PCR assay is superior to cultural methods in detecting Aspergillus spp. in murine blood samples.

ABSTRACTThe increasing incidence of invasive aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the diagnostic tools for this disease. We established a LightCycler-based real-time PCR assay to detect and quantify rapidly, specifically, and sensitivelyAspergillus fumigatusDNA in both bronchoalveolar lavage (BAL) and blood samples from high-risk patients. The primers and hybridization probes were derived from anA. fumigatus-specific sequence of the mitochondrial cytochromebgene. The assay is linear in the range between 13.2 fg and 1.3 ng ofA. fumigatusDNA, corresponding to 3 to 300,000 CFU per ml of BAL fluid or blood. No cross-amplification was observed with human DNA or with the DNA of fungal or bacterial pathogens. For clinical evaluation we investigated 10 BAL samples from nine neutropenic patients with malignant hematological diseases and 12 blood samples from seven neutropenic patients with malignant hematological diseases. Additionally, we tested one blood sample and one BAL sample from each of two neutropenic patients. In order to characterize the validity of the novel PCR assay, only samples that had shown positive results by a previously described sensitive and specific nested PCR assay were tested. Twelve of 12 BAL samples and 6 of 14 blood samples gave positive results by the LightCycler PCR assay. Eight of 14 blood samples gave negative results by the novel method. The LightCycler PCR-mediated quantification of the fungal burden showed 15 to 269,018 CFU per ml of BAL sample and 298 to 104,114 CFU per ml of blood sample. Twenty of 20 BAL samples and 50 of 50 blood samples from subjects without evidence of invasive pulmonary aspergillosis (IPA) were PCR negative. Compared to a previously described nested PCR assay, these preliminary data for the novel real-time PCR assay indicate a less sensitive rate of detection of IPA in high-risk patients, but the assay may be valuable for quantification of the fungal burden in individual clinical samples.

The incidence of systemic fungal infections in neutropenic patients with malignant hematological diseases is increasing (Beck-Sague and Jarvis 1993; Bodey, Bueltmann, et al. 1992). Patients at highest risk for these life-threatening infections are those with prolonged periods of neutropenia after intensified chemotherapy, usually patients with acute leuke-mia or after bone marrow transplantation (Denning 1998). Particularly increasing are invasive infections with Aspergillusspecies, showing high mortality rates and, if the patient survives, also considerable complications often limiting further antileukemic therapies (Denning 1998). The paragon of antifungal therapy, Amphotericin B, is toxic; chemoprophylaxis against Aspergillus species is therefore controversially discussed and not generally recommended or practiced (Schwartz, Behre et al. 1999).

ABSTRACT ETnII elements are mobile members of the repetitive early transposon family of mouse long terminal repeat (LTR) retroelements and have caused a number of mutations by inserting into genes. ETnII sequences lack retroviral genes, but the recent discovery of related MusD retroviral elements with regions similar to gag , pro , and pol suggests that MusD provides the proteins necessary for ETnII transposition in trans . For this study, we analyzed all ETnII elements in the draft sequence of the C57BL/6J genome and classified them into three subtypes (α, β, and γ) based on structural differences. We then used database searches and quantitative real-time PCR to determine the copy number and expression of ETnII and MusD elements in various mouse strains. In 7.5-day-old embryos of a mouse strain in which two mutations due to ETnII-β insertions have been identified (SELH/Bc), we detected a three- to sixfold higher level of ETnII-β and MusD transcripts than in control strains (C57BL/6J and LM/Bc). The increased ETnII transcription level can in part be attributed to a higher number of ETnII-β elements, but 70% of the MusD transcripts appear to have been derived from one or a few MusD elements that are not detectable in C57BL/6J mice. This element belongs to a young MusD subgroup with intact open reading frames and identical LTRs, suggesting that the overexpressed element(s) in SELH/Bc mice might provide the proteins for the retrotransposition of ETnII and MusD elements. We also show that ETnII is expressed up to 30-fold more than MusD, which could explain why only ETnII, but not MusD, elements have been positively identified as new insertions.

The increasing incidence of invasive aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the currently limited diagnostic tools. Using a recently developed two-step polymerase chain reaction (PCR) assay to detect 10 fg of Aspergillus DNA, corresponding to 1-5 colony-forming units (CFU)/ml of spiked samples in vitro, we prospectively examined 197 bronchoalveolar lavage (BAL) samples from 176 subjects, including 141 neutropenic, febrile patients with lung infiltrates, at risk for invasive fungal disease. Underlying diseases of these patients were haematological malignancies; 93 patients suffered from acute leukaemias. Thirty-one of these immunocompromised patients (17.6%) were PCR positive, correlating with positive BAL culture, positive histology from lung surgery or from autopsy, positive computerized tomography scans or positive galactomannan enzyme-linked immunosorbent assay. Six patients (4.3%) of this group had positive PCR results without any correlation to clinical or other diagnostic data, probably owing to contamination of the samples by ubiquitous Aspergillus spores. The samples of two patients (1.4%) with a subsequent histologically proven mould infection were PCR negative. All 102 immunocompromised patients (72.3%) with a negative PCR showed no evidence of invasive fungal disease. From 35 patients without immunodeficiency, four (11.4%) showed positive results, without evidence of invasive or non-invasive pulmonary aspergillosis. In this haematological population, the sensitivity and specificity values of the test reached 93.9% and 94.4%, the positive predictive value 83.8%, the negative predictive value 98.1%. Our data support the considerable clinical value of this PCR assay for confirming and improving diagnosis of pulmonary aspergillosis in high-risk patients.

ETn (early transposon) elements are moderate repetitive sequences present in hundreds of copies in the mouse genome. Their length ranges from 4.4 to 7.1 kb, and, like transposons, they contain long terminal repeats (LTRs) on both sides and are flanked by target site duplications (Kaghad et al. 1985). ETn-related elements can be grouped into three distinct families. Members of the ETn I and ETn II families mainly contain sequences of unknown origin in their core region. only very short stretches of retrovirus-like sequences are present, and there are no ORFs. ETn I and ETn II elements differ primarily in the 3' half of both the 5' and 3' LTR, and in the 5' end of the core region (see Fig. 1). As a consequence. only ETn II elements contain a primer binding site for tRNA(Lys). In contrast to ETn I and ETn II. members of the recently described MusD family (Mager and Freeman 2000) contain ORFs for (at least parts of) D-type virus Gag, Pro, and Pol proteins. However, in other regions they are structurally similar to ETn II elements and contain an intact primer binding site. It has been shown that MusD sequences are evolutionarily older than ETn It elements, suggesting that the latter might have arisen by recombinatory replacement of the MusD gene-coding sequences with sequences of unknown origin (Mager and Freernan 2000). ETn elements are still active as retrotransposons. In the past bears. several germ line and somatic mutations caused by fresh ETn integrations have been found (Table 1). From 19 mutations, sufficient sequence is available in seven cases to show that the insertion was an ETn II element. In eight cases, the sequence data available indicate either an ETn II or a MusD element. ETn I has not been found to be the cause of any mutations, prompting the suggestion that ETn II is the "mobile" family, whereas ETn I elements have lost the capacity to retrotranspose.

Within the recent years, novel molecular and serological methods have been developed to improve the diagnosis of invasive aspergillosis in patients with malignant hematological diseases being at highest risk for this life-threatening infection. Early diagnosis and treatment are essential for adequate therapeutic management, which, however, often remains difficult since most of the diagnostic tools used clinically at present either lack specificity or sensitivity, or both at the worst. The clinical value, advantages and remaining problems of recently developed serological assays and molecular approaches to detect this emerging pathogen are reviewed.

Bronchoalveolar lavage (BAL) samples from 67 patients who were at high risk for invasive aspergillosis were examined using a recently developed 2-step polymerase chain reaction (PCR) that detects </=10 fg of Aspergillus DNA in blood and BAL samples in vitro. Thirteen of these patients had PCR and diagnostic results positive for Aspergillus infection. Four patients with possible invasive aspergillosis also had positive PCR results, and the remaining 50 had negative PCR results. In addition, 907 blood samples from 218 high-risk patients were screened. Thirty-three patients with positive PCR results had invasive aspergillosis; 148 patients had PCR and diagnostic results that were negative, and 34 patients with positive PCR results had nonconclusive clinical data. Both blood and BAL testing were performed for 45 patients. All 8 patients with proven invasive aspergillosis showed concordance of positive PCR results. Our data suggest that this PCR method has possible clinical value for confirming and improving the diagnosis of invasive aspergillosis in high-risk patients.

Evolution over millions of years has adapted several thousand copies of retrovirus-like elements and over 10 times as many solitary long terminal repeats (LTRs) to their present location in the human genome. Transcription of these human endogenous retroviruses (HERVs) has been detected in various cells and tissues, and in some cases their transcriptional control elements have been recruited by cellular genes. We used a retroviral pol-specific expression array to obtain a HERV transcription profile in a variety of human cells such as epidermal keratinocytes, liver cells, kidney cells, pancreatic cells, lymphocytes, and lung fibroblasts. This rapid screening test revealed a distinct HERV pol-expression pattern in each cell type tested so far. About 40 different U3/R regulatory sequences from the HERV-H and HERV-W families were then amplified from actively transcribed 3'HERV LTRs of various cell lines and tissues. Their promoter activities were compared with LTR sequences of other known HERV families in 12 human cell lines using a transient luciferase reporter system. Expression of the isolated HERV LTRs varied significantly in these cell lines, in some cases showing strict cell type specificity. These results suggest that endogenous retroviral LTRs may be a valuable source of transcriptional regulatory elements for the construction of targeted retroviral expression vectors.

The human genome harbors thousands of long terminal repeats (LTRs) that are derived from endogenous retroviruses and contain elements able to regulate the expression of neighboring cellular genes. We have investigated the ability of human endogenous retroviral (HERV)-K LTRs to provide transcriptional processing signals for nonviral sequences. Four chimeric cDNA clones isolated from a cDNA library derived from the human cell line T47D were found to be polyadenylated within an HERV-K-T47D-related LTR. Two transcripts containing an as yet unknown cellular sequence were probably derived from the same genomic locus but their 3' ends were processed at different positions of the LTR. Structural analysis of the polyadenylation site suggests RNA stem-loop structures similar to the HTLV-1 Rex responsive element that bring the two remote AAUAAA and GU-rich elements into the spatial juxtaposition necessary for correct 3' end processing. The cellular part of the third chimeric clone shows significant homology to an exon of the human tyrosine phosphatase 1 gene, although oriented in the antisense direction compared to the adjacent LTR. Furthermore, we found that the 3' untranslated region of the human transmembrane tyrosine kinase gene FLT4 is probably derived from a partial HERV-K-T47D LTR sequence. Taken together, our data suggest that LTRs of the HERV-K-T47D family display biological function by mediating polyadenylation of cellular sequences.

We have developed a highly sensitive, universal assay that allows detection as well as identification of all known retroviral reverse transcriptase (RT)-related nucleic acids in a biological sample by a single two-step experiment. The assay combines polymerase chain reaction (PCR) and reverse dot-blot hybridization (RDBH), using an array of immobilized synthetic retrovirus-specific oligonucleotides and two sets of mixed oligo primers (MOPs). These primers were derived from highly conserved motifs found in all known reverse transcriptase genes. The PCR/RDBH assay was used for qualitative analyses of human endogenous retrovirus (HERV) transcription in peripheral blood mononuclear cells (PBMCs) and in particles released by the human mammary carcinoma-derived cell line T47D. Sensitivity was further demonstrated by detection of down to 10 copies of pig endogenous retrovirus (PERV) DNA in human cDNA samples. Therefore, this assay is particularly useful for the identification of retroviral sequences in xenografts as well as in recipients of xenografted tissues and organs. Moreover, it is a valuable tool to detect retroviral transcripts and particles in cell cultures used for production of therapeutic polypeptides. The assay is further suitable for monitoring vector preparation used in human gene therapy to exclude transfer of copackaged endogenous retroviruses into target cells.

A new type C retrovirus-related endogenous pol sequence (ERV-FTD) found to be occasionally copackaged in retrovirus-like particles released by the human mammary carcinoma cell line T47D was used to screen a human genomic library (Seifarth W, Skladny H, Krieg-Schneider F, Reichert A, Hehlmann R, and Leib-Mösch C: J Virol 1995;69:6408-6416). The DNA sequence of one full-length clone now reveals a human endogenous proviral sequence (HERV) of 4190 bp in length comprising a 5' LTR (489 bp) and regions with 37 and 74% overall amino acid homology to RTVL-Ia gag and pol genes, respectively. About 35 related elements were found to be distributed on all human chromosomes except 16, 17, and Y. Sequence comparisons with Mo-MuLV and various type C-related HERVs suggest that despite a proline primer-binding site this novel HERV element, now named HERV-IP-T47D, can be assigned to one family together with known HERV-I elements. Phylogenetic analyses of 5 proviral and 25 solitary LTR sequences confirmed the existence of two distinct but closely related subgroups of the HERV-IP superfamily in the primate genome. In contrast to most known HERV-families, the evolutionary age of HERV-IP elements dates back prior to the divergence of New and Old World monkeys. Despite their old age, members of the HERV-IP family are still transcriptionally active and were found to be highly expressed in specific human tissues such as liver and kidney.

ABSTRACT The increasing incidence of aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the currently limited diagnostic tools. We developed a two-step PCR assay that specifically amplifies a region of the 18S rRNA gene that is highly conserved in Aspergillus species. A number of primers with the least homology to equivalent human or Candida gene sequences were screened for the pairs that gave the highest sensitivity and specificity. No cross-reaction with the wide range of fungal and bacterial pathogens so far tested was observed. This assay allows direct and rapid detection of down to 10 fg of Aspergillus DNA corresponding to 1 to 5 CFU per ml of blood. A total of 315 blood and bronchoalveolar lavage samples from 140 subjects, including 93 patients at risk for invasive fungal disease, were screened. The result was a 100% correlation between positive histology, culture, or high-resolution computed tomography findings and PCR results. The test specificity was 89%. Our data point to the considerable potential clinical value of this simple, specific, rapid, and inexpensive PCR assay for improving the means of early diagnosis of systemic aspergillosis in high-risk patients.

ABSTRACT We previously described that type B retrovirus-like particles released from the human mammary carcinoma cell line T47D are pseudotypes and package retroviral RNA of different origins (W. Seifarth, H. Skladny, F. Krieg-Schneider, A. Reichert, R. Hehlmann, and C. Leib-Mösch, J. Virol. 69:6408–6416, 1995). One preferentially packaged retroviral sequence, ERV-MLN, has now been used to isolate the corresponding full-length provirus from a human genomic library. The 9,315-bp proviral genome comprises a complete retroviral structure except for a 3′ long terminal repeat (LTR) truncation. A lysine tRNA primer-binding site and phylogenetic analyses assign this human endogenous retroviral element, now called HERV-K-T47D, to the HML-4 subgroup of the HERV-K superfamily. The gag,prt, pol, and env genes exhibit 40 to 60% amino acid identity to HERV-K10. HERV-K-T47D is located on human chromosome 10, with five closely related elements on chromosomes 8, 9, 15, 16, and 19 and several hundred HERV-K-T47D-related solitary LTRs dispersed over the human genome. HERV-K-T47D-related sequences are detected in the genomes of higher primates and Old World monkeys but not in those of New World monkeys. High HERV-K-T47D transcription levels were observed in human placenta tissue, whereas transcription in T47D cells was strictly steroid dependent.

Retinoic acid (RA) is essential for regulation of epithelial cell differentiation. The intracellular effects of RA are mediated by RA-binding nuclear receptors, including the RA receptors (RARs) alpha, beta, and gamma. The ligand-activated receptors induce the transcription of target genes by binding to RA-responsive elements in the promoter regions. One target gene is the RAR beta gene, which encodes a potential tumor suppressor. Loss of RA inducibility of RAR beta gene expression is assumed to play a role in the development of several types of human carcinomas, including carcinomas of the uterine cervix. We have analyzed RAR beta gene expression in normal cervical cells and in cervical carcinoma cell lines. The results show that the RAR beta mRNA levels are high and RA inducible in the primary keratinocytes, whereas they are low and not inducible or only slightly inducible by RA in all of the cervical carcinoma cell lines analyzed. The basal and the RA-induced RAR beta mRNA levels tend to increase with senescence of the normal cells. Fusion of primary ectocervical keratinocytes with HeLa cervical carcinoma cells revealed that the characteristics of RAR beta gene expression of the normal cells are dominant over that of the tumor cells. Using synthetic retinoids with receptor-preferential agonist activities and a RAR alpha-specific antagonist, we show that RAR alpha is the major endogenous RAR subtype for induction of RA-dependent RAR beta gene expression. Taken together, our results indicate that abnormal downregulation of RAR beta gene expression may be an important step in the multifactorial process of cervical carcinogenesis.