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.

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.

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.

[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.

Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2.

Postweaning multisystemic wasting syndrome (PMWS) is a disease of nursery and fattening pigs characterized by growth retardation, paleness of the skin, dyspnea, and increased mortality rates. Porcine circovirus 2 (PCV2) has been demonstrated to be the cause of PMWS. However, other factors are needed for full development of the syndrome, and porcine reproductive and respiratory syndrome virus (PRRSV) infection has been suggested to be one of them. Twenty-four conventional 5-week-old pigs were distributed in four groups: control (n = 5), PRRSV inoculated (n = 5), PCV2 inoculated (n = 7), and PRRSV and PCV2 inoculated (n = 7). The two groups inoculated with PRRSV showed growth retardation. Pigs inoculated with both PRRSV and PCV2 had increased rectal temperature. One of these pigs developed wasting, had severe respiratory distress, and died. The most important microscopic lesion in pigs inoculated with PCV2 was lymphocyte depletion with histiocytic infiltration of the lymphoid organs, more severe and in a wider range of tissues in doubly inoculated pigs. Interstitial pneumonia was observed in the three inoculated groups. PCV2 nucleic acid was found by in situ hybridization in larger amounts and in a wider range of lymphoid tissues in PRRSV- and PCV2-inoculated than in PCV2-inoculated pigs. TaqMan PCR was performed to quantify the PCV2 loads in serum during the experiment. PCV2 loads were higher in doubly inoculated pigs than in pigs inoculated with PCV2 alone. These findings indicate that severe disease can be reproduced in conventional 5-week-old pigs by inoculation of PRRSV and PCV2. Moreover, these results support the hypothesis that PRRSV infection enhances PCV2 replication.

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.

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.

Human herpesvirus 6A DNA Is detected frequently in plasma but rarely in peripheral blood leukocytes of patients after bone marrow transplantation.

A real-time quantitative polymerase chain reaction assay was devised to determine the load of human herpesvirus (HHV)-6A and -6B DNA in paired samples of plasma and peripheral blood leukocytes (PBL) of 25 bone marrow transplant patients. The assay detects HHV-6 DNA variants A and B in a linear range of 10(7)-10(1) genome equivalents per assay. Viral DNA was measured in 336 paired DNA PBL samples and in corresponding plasma samples. HHV-6A and/or -6B DNA was detected in PBL of 23 of 25 patients and in plasma of 24 of 25 patients. HHV-6B was the predominant variant found in PBL and also was detected in the corresponding plasma. Surprisingly, only 1 of 25 patients had detectable HHV-6A DNA in PBL, although 23 of 25 patients were positive for HHV-6A DNA in plasma. HHV-6 DNA load in plasma was significantly higher for HHV-6A than for HHV-6B (P=.0066).

Genetics of atopy in a mouse model: polymorphism of the IL-5 receptor alpha chain.

To study the genetics of atopy systematically we established a mouse model that provides the general phenotype of atopy: the early response characteristic of IgE-dependent eczema or atopic dermatitis, and the diagnostic test of atopy, the skin-prick test. Using an immediate cutaneous hypersensitivity test (ICHS) against birch pollen extract we could classify A/J and C57BL/6 (B6) inbred mouse strains respectively as high responder and low responders. The F1 hybrids were found to be high responders with incomplete penetrance. Backcrossing F1 mice to the low responder B6 strain yielded three classes of responders, high, intermediate, and low. A genome-wide microsatellite screen of the backcross progeny disclosed suggestive linkage to a microsatellite marker on chromosome 6 close to the locus of the IL-5 receptor alpha chain. Its allelic variation in A/J and B6 strains was investigated and two major differences were detected. Firstly, a nucleotide exchange in the 5' untranslated region of B6 mRNA resulted in increased transcription/translation of a reporter construct. Higher expression of the receptor on the cell surface would be expected to favor an allergic immune response. Secondly, the two alleles are differentially spliced so as to yield two soluble isoforms in A/J mice versus one in B6 mice. Higher expression of soluble IL-5R would be expected to reduce the level of allergy through capture of IL-5. Thus both findings conform to the expectation based on susceptibility to atopy and thus identify the IL-5R alpha chain as a likely contributor to the genetics of atopy.

Detection of human cytomegalovirus DNA by real-time quantitative PCR.

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.

Applicability of an absolute quantitative procedure to monitor intra-individual bcr/abl transcript kinetics in clinical samples from chronic myelogenous leukemia patients.

Chimeric bcr/abl fusion proteins are thought to be the molecular 'pathogen' of chronic myelogenous leukaemia (CML). Expression levels of the respective fusion RNAs reflect disease progression as well as remission upon therapeutic intervention in CML patients. However, there is no quick and reliable method that would allow the quantitative routine monitoring of bcr/abl hybrid transcripts. A fluorescent probe-based PCR assay (TaqMan) has been described to quantitfy the exact amount of target sequences. We have established TaqMan real-time RT-PCRs for M-bcr/abl (b2a2, b2a3, b3a2, b3a3) and m-bcr/abl (e1a2) fusion transcripts as well as for beta-actin. All PCRs quantified as little as 10 copies/100 ng total cDNA. In order to investigate whether this procedure is appropriate for routine diagnostic monitoring, we performed retrospective measurements on 9 documented CML disease courses. Our data show that ongoing or relapsing CML is paralleled by increasing peripheral levels of bcr/abl fusion RNAs. Furthermore, sucessful anti-leukemic treatment is reflected by decreasing absolute bcr/abl transcript numbers. In contrast with conventional bcr/abl PCR techniques we could distinguish single positive values and gradually increasing copy numbers.

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.

Evidence for a post-Columbian introduction of human T-cell lymphotropic virus [type I] [corrected] in Latin America.

To investigate the origin and dissemination of human T-cell lymphotropic virus type I in Latin America, we performed phylogenetic analysis on the LTR and env sequences of 13 HTLV-I isolates from Peruvians of four different ethnic groups: blacks and some mulattos of African origin; Quechuas of Inca origin; Nikkei of Japanese descendance; and Mestizos, a mixed population of white and Indian origin. All Peruvian samples could be situated within the cosmopolitan subtype HTLV-Ia, yet one sample showed an indeterminate Western blot pattern, lacking reactivity towards the HTLV-I type specific MTA1 peptide. Within the LTR, we could confirm the previously reported subdivision into four subgroups--one big transcontinental clade A, a Japanese clade B, a West African/Caribbean clade C and a North African clade D--and we identified a new separate subgroup E of black Peruvian strains. The clustering of the Peruvian samples seemed to depend on the ethnic origin of the host. The largest heterogeneity was observed in the black Peruvian samples. The mitochondrial DNA type of one of these black Peruvian strains of subgroup E was identical to that of West African source populations of the slave trade. Both findings support the idea of multiple post-Columbian introductions of African HTLV-Ia strains into the black Latin American population. Additionally, a tight cluster of Nikkei and Japanese samples implied a separate and rather recent transmission of a Japanese lineage of HTLV-I into Peru. A well-supported cluster of Latin American strains (including Peruvian Quechuas and Colombian Amerindians) could be situated within the transcontinental group. Molecular clock analysis of the Latin American and Japanese clade resulted in an equal evolutionary rate for those strains. Along with the anthropologically documented peopling of the Americas, the analysis was more in favour of a recent (400 to 100 years ago) introduction of HTLV-Ia into the American continent rather than a Palaeolithic introduction.

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.