Gouleako virus isolated from West African mosquitoes constitutes a proposed novel genus in the family Bunyaviridae.

The family Bunyaviridae is the most diversified family of RNA viruses. We describe a novel prototypic bunyavirus, tentatively named Gouléako virus, isolated from various mosquito species trapped in Côte d'Ivoire. The S segment comprised 1,087 nucleotides (nt), the M segment 3,188 nt, and the L segment 6,358 nt, constituting the shortest bunyavirus genome known so far. The virus had shorter genome termini than phleboviruses and showed no evidence of encoded NSs and NSm proteins. An uncharacterized 105-amino-acid (aa) putative open reading frame (ORF) was detected in the S segment. Genetic equidistance to other bunyaviruses (74 to 88% aa identity) and absence of serological cross-reactivity with phleboviruses suggested a proposed novel Bunyaviridae genus.

Sequence analysis of the first HTLV-I infection in Germany without relations to endemic areas.

In most parts of Europe only a limited number of sporadic cases of HTLV-I infections have been identified. So far, the few cases found in Germany have always been linked to individuals with relations to endemic areas. Here we report the first HTLV-I infection from a German ATL patient without any known risk for HTLV-I infection and with no relations to known endemic areas. The DNA sequence of the provirus was determined, and a phylogenetic analysis based on the LTR sequence established a close relationship with HTLV-I sequences previously found in two Romanian patients. Our data suggest the existence of a previously unrecognized cluster of HTLV-I infections in southeastern or central Europe.

The HTLV type I sequence from an asymptomatic German blood donor is related to sequences from South America.

In most parts of Europe only a limited number of sporadic cases of HTLV-I infections have been identified. So far, the few cases found in Germany were individuals from endemic areas or with relations to endemic areas. Here we report an HTLV-I infection from an asymptomatic female German blood donor whose only known potential risk was a former partner from South America, where HTLV-I is known to be endemic. The DNA sequence of the LTR region was determined and a phylogenetic analysis indeed suggested homologies with HTLV-I sequences from South America.

Quantitative detection of Toxoplasma gondii DNA in human body fluids by TaqMan polymerase chain reaction.

OBJECTIVE: A new quantitative polymerase chain reaction (real-time PCR) was designed to detect Toxoplasma DNA in human body fluid samples. METHODS: Real-time fluorescence detection of amplification product formation on the basis of the TaqMan-System was established with Toxoplasma 18S rDNA as a target gene. RESULTS: The method provides a high sensitivity comparable to conventional nested PCR procedures and generates quantitative data when detecting toxoplasmic DNA in human blood, cerebrospinal or amniotic fluid. Moreover, data were obtained investigating blood samples from an immunocompromised patient with reactivated toxoplasmosis after allogeneic bone marrow transplantation, monitoring the therapeutic effect. CONCLUSIONS: The potential application of this method to detect Toxoplasma DNA in body fluids and to follow the development of parasitemia under therapy could be demonstrated.

The pathology of experimental poxvirus infection in common marmosets (Callithrix jacchus): further characterization of a new primate model for orthopoxvirus infections.

Zoonotic orthopoxvirus (OPV) can induce severe disease in man and the virus has potential for use in bioterrorism. New vaccines and therapeutics against OPV infections must be tested in animal models. The aim of this study was to characterize the clinical course and pathology of a new OPV isolate, calpox virus, which is infectious in marmosets. Infection experiments were performed with 28 common marmosets (Callithrix jacchus) exposed to different challenge doses of calpox virus by the intravenous, oropharyngeal and intranasal (IN) routes. The median marmoset IN infectious dose corresponded to 8.3 × 10(2)plaque forming units of calpox virus. Infected animals developed reproducible clinical signs and died within 4-15 days post infection. Characteristic pox-like lesions developed in affected organs, particularly in the skin, mucous membranes, lymph nodes, liver and spleen. Calpox virus disease progression and pathological findings in the common marmoset appear to be consistent with lethal OPV infections in man and in other non-human primate (NHP) models. IN inoculation with low virus doses mimics the natural route of the human variola virus infection. Thus, the marmoset model of calpox virus infection can be considered to be relevant to investigation of the mechanisms of OPV pathogenesis and pathology and for the evaluation of new vaccines and antiviral therapies.

Examining landscape factors influencing relative distribution of mosquito genera and frequency of virus infection.

Mosquito-borne infections cause some of the most debilitating human diseases, including yellow fever and malaria, yet we lack an understanding of how disease risk scales with human-driven habitat changes. We present an approach to study variation in mosquito distribution and concomitant viral infections on the landscape level. In a pilot study we analyzed mosquito distribution along a 10-km transect of a West African rainforest area, which included primary forest, secondary forest, plantations, and human settlements. Variation was observed in the abundance of Anopheles, Aedes, Culex, and Uranotaenia mosquitoes between the different habitat types. Screening of trapped mosquitoes from the different habitats led to the isolation of five uncharacterized viruses of the families Bunyaviridae, Coronaviridae, Flaviviridae, and Rhabdoviridae, as well as an unclassified virus. Polymerase chain reaction screening for these five viruses in individual mosquitoes indicated a trend toward infection with specific viruses in specific mosquito genera that differed by habitat. Based on these initial analyses, we believe that further work is indicated to investigate the impact of anthropogenic landscape changes on mosquito distribution and accompanying arbovirus infection.

Human T-cell lymphotropic virus type 1 provirus and phylogenetic analysis in patients with mycosis fungoides and their family relatives.

BACKGROUND: Mycosis fungoides (MF) is a cutaneous T-cell lymphoma of unknown aetiology. A pathogenic role of human T-cell lymphotropic virus type 1 (HTLV-1) has been suggested but remains controversial. To determine whether MF is linked to HTLV-1. METHODS: Blood samples were collected from 60 patients, 15 family relatives of patients with MF (MFRs), 20 healthy controls and 10 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The presence of HTLV-1 antibodies in serum was tested by the Western blot rp21e-enhanced test. DNA was extracted from the blood with the Qiagen blood kit. We used 500 ng of DNA either in conventional HTLV-1-specific polymerase chain reaction (PCR) or in real-time PCR using primers sk43 and sk44 together with a tax-specific fluorescent probe. RESULTS: In Western blot, antibodies against three to four HTLV-1 antigens were detected in 52% of patients with MF. All of the patients with HAM/TSP were positive, while only 7% of the MFRs and none of the 20 healthy controls reacted with HTLV-1 antigens in Western blot. One of 60 patients with MF and one of 15 MFRs were positive in HTLV-1 PCR. These two PCR-positive samples which were quantified in real-time PCR showed that fewer than five in 10(6) cells were HTLV-1 infected. We succeeded in amplifying and sequencing the 5' end of the provirus from the blood of the PCR-positive MFR by seminested PCR. A positive result was also obtained in this test. Phylogenetic tree analyses revealed a high homology of this sequence with other HTLV-1 sequences from the Middle East. The above PCR-positive MFR was the brother of a PCR-negative patient with MF. CONCLUSIONS: These findings demonstrate that HTLV-1 is probably not the aetiological agent of MF. However, it may play a role in immunosuppression and in the spreading of the disease.

Fatal poxvirus outbreak in a colony of New World monkeys.

An epizootic infection was observed in a colony of 80 New World monkeys consisting of various species including a group of marmosets and Saguinus species. During the summer and autumn of 2002, 30 animals died of unknown diseases. Six animals were sent to the German Primate Center for investigation of the cause of death. A complete pathologic and histologic investigation was carried out. The animals exhibited erosive-ulcerative lesions of the oral mucous membranes. Advanced stages of the disease were characterised by hemorrhagic lesions on the skin distributed randomly over the body, but principally on the face, scrotal region, soles, and palms. Electron microscopy revealed virus particles with orthopox-like morphology within intracytoplasmic inclusions in epithelial cells. The DNA samples from various tissues were analyzed by use of a set of orthopox virus-specific, real-time polymerase chain reaction assays. Amplification products were sequenced to define the virus more precisely. Sequencing confirmed the presence of an orthopox virus. Sequence data indicated that all six animals were infected with the same virus. Propagation of the virus on Vero cells resulted in a rapidly progressive cytopathogenic effect. Preliminary phylogenetic analyses of two genes revealed closest homology to cowpox viruses. The origin of this poxvirus outbreak remains unexplained, and the strain and genus of the virus need to be determined in detail.

[Recurrent strongyloidiasis as an indicator of HTLV-1 infection]. / Rezidivierende Strongyloidose als Indikator einer HTLV-1-Infektion.

HISTORY AND CLINICAL FINDINGS: A 53-year-old West African man presented two years after a travel to Guinea because of severe headache, neck stiffnes, fever and pruritus. The patient had been in orthopedical treatment for the last five months. INVESTIGATIONS: Stool microscopy revealed a high number of Strongyloides stercoralis larvae. Hematology, biochemistry and all other parasitology results were normal. HIV-1/2 testing was negative and CD4+-lymphocyte count was normal. Concomitant infection by Human T Cell lymphotropic virus type 1 (HTLV-1) was confirmed by serology and PCR. The phylogenetic analysis confirmed African origin of the virus. TREATMENT: The infection responded to a five-day course of albendazol at 400 mg/d but during the following five years repeat recrudescences were observed inspite of high-dosage and prolonged antiparasitic treatments. Eventually, eradication of the infection was achieved by a four day course of ivermectin 0.2 mg/kg/d. CONCLUSIONS: Although both strongyloidiasis and HTLV-1 infections occur most frequently in tropical areas, these may also be observed in temperate regions. Suppression of the immune system by HTLV-1 differs from that by HIV. CD4+-lymphocytes were rarely decreased. Prolonged treatment with ivermectin in a dosage exceeding the current recommendations may be required in HTLV-1 infected patients and was well tolerated. The unusual presentation of the infection with muscular symptoms contributed to the delay of the diagnosis. HTLV-1 positive patients must be monitored for years. They and their partners must be instructed how to prevent transmission of the virus.

Penicillin-degrading activities of peptides from pneumococcal penicillin-binding proteins.

Trypsin treatment of native penicillin-binding proteins (PBPs) 1a, 2b and 3 from Streptococcus pneumoniae resulted in the formation of stable peptides containing the beta-lactam-binding site with molecular masses ranging from 26 kDa to 36 kDa. Whereas the PBP 1a peptide (Ia) was enzymatically rather unstable, the PBP 2b peptide (IIb) and the PBP 3 peptide (III) were able to bind and release beta-lactams with similar rates compared to the intact PBP, the turnover rate of fragment II b was even twice as fast as that observed with PBP 2b. Analysis of the turnover products by thin-layer chromatography revealed that PBP 2b and 3 produced penicilloic acid as well as phenylacetylglycine. On the other hand, with the corresponding tryptic fragments only the hydrolysis product penicilloic acid was obtained.

Penicillin-binding proteins of Streptococcus pneumoniae: characterization of tryptic peptides containing the beta-lactam-binding site.

Penicillin-binding proteins of Streptococcus pneumoniae were labeled with [3H] propionyl-ampicillin and treated with trypsin. The fragments were separated on sodium dodecyl sulfate/polyacrylamide gels, and peptides containing the beta-lactam-binding site visualized by fluorography. From native penicillin-binding proteins (PBP), either membrane-bound or solubilized with Triton X-100, relatively stable end products of proteolysis were obtained. The smallest radioactive peptides from PBP 1a (92 kDa), PBP 2b (77 kDa), and PBP 3 (43 kDa ) had sizes of 36.5 kDa, 26 kDa, and 29 kDa, respectively. When the PBP were trypsin treated prior to labeling with the radioactive beta-lactam, these small peptides were still able to bind the antibiotic. Under conditions of limited proteolysis, membrane-bound PBP 2b and PBP 3 were converted into soluble, hydrophilic derivatives after loss of a peptide of only 2 kDa and 1.5 kDa, respectively. These two PBP are therefore anchored in the membrane by a small terminal peptide. In contrast, PBP 1a could be digested to a Mr of 48000 without becoming water-soluble; the only hydrophilic tryptic peptide that could be found was the 36.5 kDa fragment. Therefore, large domains of this PBP seem to be embedded in the membrane.

Antibodies against the benzylpenicilloyl moiety as a probe for penicillin-binding proteins.

Antibodies against the benzylpenicilloyl determinant were used to identify complexes of benzylpenicilloyl and penicillin binding protein (PBP) of several bacterial species on immunoblots. Since radioactive penicillin was not needed, this technique readily allowed in vivo labeling studies even in Escherichia coli, where the saturating concentration was around 0.6 mg/ml. The antibodies showed no substantial cross-reactivity to other beta-lactam-PBP complexes with the exception of 6-aminopenicillanic acid. Surprisingly, some penicilloyl-PBP were hardly recognized by the antiserum, whereas the others could be stained according to the amount of penicillin bound.

Functional tolerance of the human immunodeficiency virus type 1 envelope signal peptide to mutations in the amino-terminal and hydrophobic regions.

We demonstrated that the leader sequence of the human immunodeficiency virus type 1 envelope functions as signal peptide (SP) despite low scoring in a prediction program. As expected for SP, the hydrophobic core (HC) is essential, and no other sequence could compensate for HC deletion. Contrary to other SPs, major substitutions in the HC, such as introduction of basic, polar, or alpha-helix-breaking residues, still allowed efficient translocation and glycosylation. Also, extensive deletions or substitutions of the charged residues at the N terminus had little if any inhibitory effect. This report, which is the first study of human immunodeficiency virus SP, describes the exceptional tolerance of this peptide to mutations.

Defective human T-cell leukaemia virus type 1 (HTLV-1) genomes: no evidence in serologically indeterminate german blood donors but new type detected in established cell lines.

Individuals reactive in antibody screening tests (ELISA) and with one or more reactions to HTLV-1 proteins on Western blotting, but lacking the criteria of a confirmed HTLV infection, are not exceptional in regions with a low prevalence of HTLV-1/-2 infections. PCR analysis of these indeterminate samples, using "diagnostic" pol and tax sets of primers, give negative results. However, expression of HTLV-1 defective proviruses with internal deletions undetectable by PCR with diagnostic primers could have taken place. Seven German HTLV-1 ELISA-reactive blood donors, who showed reactivity also in Western blots against several viral proteins, and twenty haemophiliacs, were examined by nested PCR and/or PCR/Southern hybridisation with primers designed for detection of HTLV-1 defective proviruses. No HTLV-1-specific amplification products were obtained. However, HTLV-1 defective proviruses with large internal deletions were detected in four out of five cell lines established from symptomatic HTLV-1 cases and two in HUT-102 cells. In two amplicons, short inverted rRNA sequences between gag and env fragments of HTLV-1 defective proviruses were revealed. These results do not exclude the presence of defective HTLV-1 proviruses in individuals with indeterminate serology although this is unlikely.

Influence of transmembrane domains on the fusogenic abilities of human and murine leukemia retrovirus envelopes.

The envelopes of two highly divergent oncoviruses, human T-cell leukemia virus type 1 (HTLV-1) and Friend murine leukemia virus (F-MuLV), have distinct patterns of cellular receptor recognition, fusion, and syncytium formation. To analyze the influence of the transmembrane envelope subunit (TM) on fusogenic properties, we substituted either the entire TM or distinct domains from F-MuLV for the corresponding domains in the HTLV-1 envelope. Parental, chimeric, and truncated envelopes cloned into a eukaryotic expression vector were monitored for fusogenic potential in human, rat, and murine indicator cell lines by using a quantitative assay. This highly sensitive assay allowed us to assess the fusogenic properties and syncytium-forming abilities of the HTLV-1 envelope in murine NIH 3T3 cells. All chimeric envelopes containing extracellular sequences of the F-MuLV TM were blocked in their maturation process. Although deletions of the HTLV-1 cytoplasmic domain, alone and in combination with the membrane-spanning domain, did not prevent envelope cell surface expression, they impaired and suppressed fusogenic properties, respectively. In contrast, envelopes carrying substitutions of membrane-spanning and cytoplasmic domains were highly fusogenic. Our results indicate that these two domains in F-MuLV and HTLV-1 constitute structural entities with similar fusogenic properties. However, in the absence of a cytoplasmic domain, the F-MuLV membrane-spanning domain appeared to confer weaker fusogenic properties than the HTLV-1 membrane-spanning domain.

The envelopes of two ecotropic murine leukemia viruses display distinct efficiencies in retroviral vaccination by interference.

In cell cultures infected with a retrovirus, the expression of the viral envelope interferes with superinfection by retroviruses which recognize the same receptor. We have previously demonstrated that vaccination of susceptible strains of mice (of the Mus musculus species) with the attenuated ecotropic Friend murine leukemia virus (F-MuLV) B3 efficiently protects against the early hemolytic anemia and the erythroleukemia induced by a challenge with the virulent F-MuLV 57 through a similar in vivo mechanism of interference to superinfection (A. Corbin and M. Sitbon, J. Virol. 67, 5146-5152, 1993). Vaccination with the heterologous ecotropic Moloney-MuLV (M-MuLV) efficiently protects against the early hemolytic anemia but has a weak protective effect on the F-MuLV 57-induced erythroleukemia. Furthermore, vaccination with the attenuated F-MuLV B3 had only a transient protective effect on M-MuLV-induced thymomas. These different efficiencies of F- and M-MuLV to confer protection in this model of vaccination by interference were mostly due to envelope sequences, indicative of distinct in vivo interference properties of the two ecotropic envelopes.

[Quantitative determination of CMV-DNA in saliva of patients with bone marrow and stem cell transplantation using TaqMan-PCR]. / Quantitative Bestimmung humaner CMV-DNA im Speichel von Patienten mit Knochenmark- und Stammzelltransplantation mithilfe der TaqMan(R)-PCR.

BACKGROUND: Human cytomegalovirus (HCMV) infection is associated with severe and life-threatening diseases in immunocompromised patients, especially after bone marrow (BM) and stem cell (SC) transplantation. Prior to transplantation the potential risk of HCMV disease is therefore determined by HCMV-antibody blood testing of transplant donor (D) and recipient (R). Virus carriers are positive for anti-CMV-IgG. Virus patterns are distinguished as follows: group 1 (D+/R+), group 2 (D-/R+), group 3 (D+/R-), and group 4 (D-/R-). AIM: The aim of this study was qualitative and quantitative determination of the HCMV DNA load in saliva of BM and SC transplantation patients. PATIENTS AND METHOD: Unstimulated saliva was collected from 20 patients prior to BM and SC transplantation, during the time of conditioning, and after transplantation. DNA was isolated and analyzed for evidence of HCMV DNA with TaqMan PCR. RESULTS: HCMV DNA was isolated in seven cases. In all group 1 patients (D+/R+) HCMV DNA could be demonstrated. Only three of seven group 2 patients (D-/R+) were positive for HCMV DNA. The only group 3 patient (D+/R-) and all eight group 4 patients (D-/R-) were negative. CONCLUSION: TaqMan PCR is a reliable method for HCMV DNA quantification. In three patients (anti-HCMV-IgG positive) who received an anti-CMV-IgG negative transplant HCMV DNA was isolated. In contrast, no HCMV-DNA was evident in HCMV-negative patients who received an HCMV-negative transplant. Accordingly, the risk of HCMV reactivation is more probable than the risk of reinfection.