On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria.

This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/µL, 1 GU/µL, and 5 × 10(3) GU/µL (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches.

Late cytomegalovirus polyradiculopathy following haploidentical CD34+-selected hematopoietic stem cell transplantation.

A 55-year-old man with acute myeloid leukemia in second relapse presented 4 months after haploidentical CD34+-selected hematopoietic stem cell transplantation (HSCT) with symmetric, progressive neurological deficits of the lower extremities. Although there was no molecular evidence for drug resistance in the cerebral-spinal fluid, antiviral combination therapy failed to control the rapidly progressing CMV polyradiculopathy (PRP) and encephalitis, which were confirmed by autopsy studies. Late CMV PRP as an unusual manifestation of CMV disease should be kept in mind in patients with suggestive neurological symptoms after HSCT.

Molecular epidemiology of hepatitis B virus variants in nonhuman primates.

We characterized hepatitis B virus (HBV) isolates from sera of 21 hepatitis B virus surface antigen-positive apes, members of the families Pongidae and Hylobatidae (19 gibbon spp., 1 chimpanzee, and 1 gorilla). Sera originate from German, French, Thai, and Vietnamese primate-keeping institutions. To estimate the phylogenetic relationships, we sequenced two genomic regions, one located within the pre-S1/pre-S2 region and one including parts of the polymerase and the X protein open reading frames. By comparison with published human and ape HBV isolates, the sequences could be classified into six genomic groups. Four of these represented new genomic groups of gibbon HBV variants. The gorilla HBV isolate was distantly related to the chimpanzee isolate described previously. To confirm these findings, the complete HBV genome from representatives of each genomic group was sequenced. The HBV isolates from gibbons living in different regions of Thailand and Vietnam could be classified into four different phylogenetically distinct genomic groups. The same genomic groups were found in animals from European zoos. Therefore, the HBV infections of these apes might have been introduced into European primate-keeping facilities by direct import of already infected animals from different regions in Thailand. Taken together, our data suggest that HBV infections are indigenous in the different apes. One event involving transmission between human and nonhuman primates in the Old World of a common ancestor of human HBV genotypes A to E and the ape HBV variants might have occurred.

Variation within the glycoprotein B gene of human cytomegalovirus is due to homologous recombination.

Human cytomegalovirus (HCMV) strains can be classified into different glycoprotein B (gB) genotypes. In a previous study, frequent intragenic variation of the gB gene was shown. The aim of this study was to analyse whether gB variation was due to homologous recombination. The gB gene of DNA extracts derived from the peripheral blood leukocytes of 14 immunosuppressed patients was amplified by PCR and cloned. Three variable sites of gB were analysed by restriction fragment analysis and DNA sequencing and compared with published prototypic strains. In three patients doubly infected with two distinct HCMV gB strains, prototypic (60-85%) and non-prototypic recombinant strains (5-40%) were detected. To demonstrate that homologous recombination is driving HCMV gB variability, cells were coinfected with plaque-purified prototypic gB strains and recombinant gB genes were selectively amplified by PCR. gB recombinants were detected after 15 days of coculture and cross-over sites were determined by sequencing. These data indicate that homologous recombination contributes to the variability of the gB gene in vitro and in vivo.

Intragenic variability of human cytomegalovirus glycoprotein B in clinical strains.

Human cytomegalovirus (HCMV) strains can be classified into four glycoprotein B (gB) genotypes, and there has been evidence of differences in viral virulence. In this study, intragenic variability of HCMV gB strains was analyzed. The gB gene was amplified by nested polymerase chain reaction using samples from immunosuppressed patients. The genotype of fragments corresponding to the cleavage site of gB was determined by restriction fragment analysis; fragments corresponding to the N- and C-termini (gBn and gBc) were sequenced and compared with published sequences. At the cleavage site, the four known genotypes were found. Typing revealed four major genotypes at the N-terminus and two at the C-terminus. In 22 of 44 strains, the gB type determined at the cleavage site was different from the gBn or gBc type (or either), indicating that intragenic variability within the gB gene occurs frequently.

Glycoprotein B genotype correlates with cell tropism in vivo of human cytomegalovirus infection.

Human cytomegalovirus (HCMV) strains can be classified into four genotypes of the glycoprotein B (gB). In a previous study, the gB genotype 1 was found more frequently in bone marrow transplant recipients with nonfatal HCMV infection than in patients who died from HCMV disease [Fries et al. (1994): Journal of Infectious Diseases 169:769-774]. The distribution and cell tropism of different gB types in vivo were investigated. The gB type of HCMV was determined in blood or urine specimen from 76 organ and 47 bone marrow transplant recipients using PCR and restriction fragment length polymorphism (RFLP). The leukocyte populations (polymorphonuclear leukocytes, monocytes, T lymphocytes, non-T lymphocytes) of 20 viremic patients were purified by a fluorescence-activated cell sorter (FACS) and examined for HCMV infection by PCR. Sequence analysis of four randomly selected strains showed that gB types were similar to published sequences and no atypical gB types were found. Within the compartments blood and urine, the gB types were almost equally distributed, whereas the gB type 1, in contrast to gB types 2 and 3, did not infect T lymphocytes in vivo. These data show that the gB type correlates with viral tropism in vivo and thus provides further evidence that the gB variation may indeed influence the virulence of HCMV.

Infection of blood and bone marrow cells with the human cytomegalovirus in vivo.

The human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. Both, primary infection and reactivation of latent virus can cause disease. Peripheral blood leukocytes (PBL) most likely play an important role in viral persistence and dissemination of infection. However, an open question has been whether HCMV actively replicates in PBL in vivo and whether the progenitor cells in the bone marrow are also infected. Previous studies on this issue are controversial. Here we summarize data on the tropism of HCMV for mature leukocyte populations as well as bone marrow progenitor cells during HCMV viremia. All cell populations were highly purified by a fluorescence activated cell sorter (FACS) and analyzed by PCR for the presence of viral genomic DNA. Moreover, mature leukocyte populations were investigated for mRNA expression of regulatory and viral structural proteins. We could show, that HCMV DNA was detected most frequently in granulocytes and monocytes as well as in CD34+ progenitor cells of immunosuppressed patients. Viral mRNA expression was found in granulocytes, monocytes, and lymphocyte fractions. In contrast, no HCMV DNA was found in healthy, seropositive individuals.

Human cytomegalovirus glycoprotein B genotypes in renal transplant recipients.

Based on sequence variation of the glycoprotein B (gB) gene, human cytomegalovirus (HCMV) strains can be classified into four gB genotypes. In a previous study of bone marrow transplant recipients, infection with the gB type 1 correlated with a more favorable clinical outcome than infection with the gB types 2, 3, or 4. The gB type was determined in 60 renal transplant and in 47 bone marrow transplant recipients using PCR and restriction analysis. All HCMV variants in patient specimens could be assigned to one of the four previously described gB types. Two or more specimens obtained from 39 patients were analysed; in 31 of these patients the gB type was the same in all samples. The gB type did not correlate with the clinical outcome or the level of viremia in renal transplant recipients.

Glycoprotein B genotype of human cytomegalovirus: distribution in HIV-infected patients.

Glycoprotein B (gB) is involved in cell to cell transmission of human cytomegalovirus (HCMV) and may be a critical factor in tissue tropism and viral pathogenesis. We analyzed the distribution of the four known gB genotypes of HCMV in 99 HIV-positive patients. 29 patients had HCMV retinitis, and 70 patients had asymptomatic HCMV infection. DNA was isolated from blood, urine, and aqueous humor, and gB genotypes were determined by PCR and restriction analysis. Infections with gB type 1 were less frequent in patients with retinitis than in patients with asymptomatic HCMV infection (17% versus 37%; p = 0.05). Furthermore, the gB type was correlated with dissemination of infection. In patients with HCMV detected in only one compartment (blood or urine) the gB type 1 was found more frequently than in patients with HCMV detected in at least two compartments (p = 0.01). The data show that gB genotypes differ in their association with clinical disease, and indicate that the gB genotype may contribute to the course of HCMV infection.

Detection of cytomegalovirus DNA in CD34+ cells from blood and bone marrow.

Infection of hematopoietic progenitor cells with the human cytomegalovirus (HCMV) has been proposed as an explanation for the cytopenias associated with HCMV-related disease. To test this hypothesis, CD34+ cells, which include the hematopoietic progenitors, as well as mature leukocyte populations were purified on a fluorescence-activated cell sorter and analyzed for HCMV DNA by polymerase chain reaction (PCR). A total of 33 samples from 31 immunosuppressed as well as immunocompetent HCMV-seropositive individuals were studied. CD34+ cells were PCR-positive in four of seven bone marrow aspirates from allogeneic bone marrow transplant recipients, in three of eight aspirates from patients with acquired immunodeficiency syndrome, and in the first of two bone marrow samples from an immunocompetent patient with primary HCMV disease. CD34+ cells purified from peripheral blood for autologous and allogeneic transplantation were also analyzed, and 4 of 13 samples were HCMV DNA-positive. Interestingly, two of the four HCMV-positive samples were from healthy allogeneic donors. Among the mature leukocyte populations, the monocytes were most frequently found to be HCMV DNA-positive. No HCMV DNA was detected in the total bone marrow leukocytes of 13 healthy seropositive bone marrow donors or in the CD34+ cell fraction of three further seropositive donors. In conclusion, the data provide strong evidence that CD34+ hematopoietic progenitor cells can be infected with HCMV in immunosuppressed patients, while this cell population was not identified as a major viral reservoir in healthy HCMV-seropositive individuals.

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.

CD34+ hematopoietic progenitor cells are not a major reservoir of the human immunodeficiency virus.

Hematologic abnormalities occur in the majority of patients with acquired immunodeficiency syndrome (AIDS). Infection of the hematopoietic progenitor cells has been proposed as a potential explanation. In this study, different bone marrow cell populations, including the CD34+ hematopoietic progenitor cells, were purified by a fluorescence-activated cell sorter (FACS) and analyzed for the presence of human immunodeficiency virus-1 (HIV-1) proviral DNA using the polymerase chain reaction. A group of 14 patients with AIDS or AIDS-related complex (ARC) was studied (11 with peripheral blood cytopenias). The CD4+ helper cells in the bone marrow were found positive for HIV-1 DNA in all patients. In contrast, CD34+ progenitor cells were positive in only one patient. Two monocyte samples and two samples of CD4-/CD34- lymphocytes/blasts (mainly B and CD8 lymphocytes) were positive. Proviral DNA could not be detected in granulocytes. FACS analysis showed that the percentage of CD34+ hematopoietic progenitor cells was not altered in the bone marrow of AIDS patients in comparison with the HIV-1 seronegative controls. In contrast, the number of CD4+ lymphocytes was markedly reduced in the bone marrow of AIDS patients. These results show that the hematologic abnormalities in AIDS patients are neither explained by direct infection of the hematopoietic progenitor cells with HIV-1 nor by a depletion of progenitor cells.

Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies.

Monoclonal antibodies with differing specificity were prepared against the Josiah strain of the Lassa virus. All monoclonal antibodies were characterized by subclass determination and the immunofluorescence test against Lassa, LCM (WE & Arm strain), Junin, Machupo, and other arenavirus antigens. In radioimmune precipitation tests using purified Lassa virus antigen all monoclonal antibodies precipitated a single band of 60 kd, specific for the viral nucleoprotein (p 60). Three domains (A, B, C) were identified on the surface of the Lassa virus nucleoprotein using an ELISA-inhibition test. All domains carried different Lassa virus specific epitopes. In addition, the A-domain carried a group specific epitope present within the arenavirus family as a whole as shown by cross-reaction in immunofluorescence tests. The B-domain only carries Lassa virus specific epitopes, whereas the C-domain has a type specific and a subgroup specific (Lassa, LCM) epitope.