Cornelia Henke-Gendo

Activation of Mitogen-Activated Protein Kinase and NF-κB Pathways by a Kaposi's Sarcoma-Associated Herpesvirus K15 Membrane Protein

ABSTRACT The K15 gene of Kaposis sarcoma-associated herpesvirus (also known as human herpesvirus 8) consists of eight alternatively spliced exons and has been predicted to encode membrane proteins with a variable number of transmembrane regions and a common C-terminal cytoplasmic domain with putative binding sites for SH2 and SH3 domains, as well as for tumor necrosis factor receptor-associated factors. These features are reminiscent of the latent membrane proteins LMP-1 and LMP2A of Epstein-Barr virus and, more distantly, of the STP, Tip, and Tio proteins of the related γ2-herpesviruses herpesvirus saimiri and herpesvirus ateles. These viral membrane proteins can activate a number of intracellular signaling pathways. We have therefore examined the abilities of different K15-encoded proteins to initiate intracellular signaling. We found that a 45-kDa K15 protein derived from all eight K15 exons and containing 12 predicted transmembrane domains in addition to the cytoplasmic domain activated the Ras/mitogen-activated protein kinase (MAPK) and NF-κB pathways, as well as (more weakly) the c-Jun N-terminal kinase/SAPK pathway. Activation of the MAPK and NF-κB pathways required phosphorylation of tyrosine residue 481 within a putative SH2-binding site (YEEVL). This motif was phosphorylated by the tyrosine kinases Src, Lck, Yes, Hck, and Fyn. The region containing the YEEVL motif interacted with tumor necrosis factor receptor-associated factor 2 (TRAF-2), and a dominant negative TRAF-2 mutant inhibited the K15-mediated activation of the Ras/MAPK pathway, suggesting the involvement of TRAF-2 in the initiation of these signaling routes. In contrast, several smaller K15 protein isoforms activated these pathways only weakly. All of the K15 isoforms tested were, however, localized in lipid rafts, suggesting that incorporation into lipid rafts is not sufficient to initiate signaling. Additional regions of K15, located presumably in exons 2 to 5, may therefore contribute to the activation of these pathways. These findings illustrate that the 45-kDa K15 protein engages pathways similar to LMP1, LMP2A, STP, Tip, and Tio but combines functional features that are separated between LMP1 and LMP2A or STP and Tip.

Varicella vaccination in a child with acute lymphoblastic leukaemia

In July, 2003, during reinduction treatment 5 months after diagnosis of acute lymphoblastic leukaemia (ALL), a 4-yearold girl presented with generalised tonic-clonic seizures. She had been treated according to protocol ALL-BFM 2000. Cranial CT and analysis of cerebrospinal fl uid showed no signs of cerebral haemorrhage. Ultra sonography showed an enlarged liver and no signs of ascites or veno-occlusive disease. Her skin appeared normal, with no vesicular rashes. Blood tests showed only raised concentrations of aminotransferases. During the next few hours, she developed respiratory insuffi ciency, petechiae, haematomas, and vesicular lesions of the oral and vagi nal mucosal. On the assumption of an underlying infec tious cause, intravenous treatment with piperacillin, sulbactam, tobramycin, IgG, and aciclovir was initiated. 48 h after the fi rst seizure, her laboratory test results deteriorated, with aspartate and alanine aminotransferase concentrations increasing to 20 864 U/L and 16 029 U/L, respectively, and the full blood cell count indicated pancytopenia. Within 12 h, she developed multi-organ failure (liver, renal, and circulatory failure, and acute respiratory distress syndrome [ARDS]), necessitating artifi cial ventilation. Serostatus for varicella-zoster virus (VZV) was negative, but PCR for VZV was positive in peripheral blood samples (7×106 genome copies per mL). VZV was also isolated from a nasopharyngeal swab but not from cerebrospinal fl uid. PCR analysis of peripheral blood was negative for hepatitis B and C viruses, herpes simplex virus 1 and 2, Epstein-Barr virus, cytomegalovirus, adenovirus, enterovirus, human herpes virus 6, and parvovirus B19. High doses of VZVIgG were added to the treatment. Despite haemo dialysis and ventilation, the child died of progressive ARDS and multi-organ failure 10 days after admission. On receiving the positive VZV-PCR results, the mother recalled that her daughter had received live attenuated VZV vaccine (Varilrix) at another hospital 32 days before the onset of symptoms. Partial sequencing of VZV genes 38 and 54 isolated from the patient excluded a wild-type VZV infection and showed that viraemia was caused by the VZV vaccine strain OKA (fi gure). Vaccination was done 5 months after complete remission had been achieved; at that time lymphocyte count was more than 1·5×109/L, and chemotherapy was interrupted for 1 week before and after vaccination. Deaths after vaccinations with numerous attenuated viruses are well established. Fatal wild-type VZV infections have been reported in ALL patients during chemotherapy and after bone-marrow cell transplantation. Therefore, VZV vaccination is a useful, and generally accepted, therapeutic measure for patients with ALL in remission. Studies of VZV vaccination 3–4 months after autologous stem-cell transplantation, and in early ALL maintenance therapy, did not show fatal side-eff ects. However, any interruption of maintenance therapy in ALL can adversely aff ect outcome for the patient. In our patient, liver failure developed 5 weeks after VZV vaccination, which indicates longstanding replication of OKA strain in the liver. This suggestion accords with observations of late onset of complications (fever, vesicles, and severe hepatitis) in immunocompromised patients after VZV vaccination. Therefore, although we cannot fully exclude that intensifi cation of chemotherapy could have aggravated her symptoms, we suggest that VZV vaccination in seronegative children with leukaemia, who are in complete remission for at least 12 months, should not be undertaken until at least 9 months after the end of immunosuppressive treatment (including maintenance therapy) and not before a lymphocyte count of at least 1·5×109/L has been ascertained. In addition, high-risk patients should remain under close surveillance in the critical phase (6 weeks after vaccination) so that immediate antiviral treatment with aciclovir can be initiated in symptomatic children.

New Real-Time PCR Detects Prolonged Norovirus Excretion in Highly Immunosuppressed Patients and Children

ABSTRACT Noroviruses (NoV) are a major cause of epidemic nonbacterial gastroenteritis and affect all age groups worldwide. Three of five NoV genogroups, namely, genogroup I (GI), GII, and GIV, are associated with human disease. Unfortunately, these genogroups demonstrate a high degree of sequence diversity, complicating the design of pan-NoV diagnostic PCR tests. To decrease the risk of false-negative test results, we have developed a new one-step real-time TaqMan reverse transcription-PCR protocol. This protocol detects all human NoV genogroups in one reaction with a sensitivity of 400 virus genome equivalents/reaction for both GI and GII. The use of in vitro-transcribed NoV RNA as an external standard allows (semi)quantification of viral loads in samples. In a retrospective analysis of 206 stool samples from 77 patient episodes, the duration of NoV excretion and the amount of virus excreted were determined. Twenty (26.0%) of these episodes lasted longer than 10 days. Univariate risk factor analysis revealed the patient status after organ transplantation (odds ratio [OR], 7.49 [95% confidence interval, 2.06 to 28.32]; P < 0.001), immunosuppression (OR, 9.19 [95% confidence interval, 2.50 to 35.39]; P < 0.001), and age of less than 10 years (OR, 4.58 [95% confidence interval, 1.36 to 15.77]; P = 0.004) as risk factors for a NoV excretion period of more than 10 days. These findings were confirmed by time-dependent Kaplan-Meier analyses, whereas multivariate Cox regression analyses identified immunosuppression as the sole risk factor. Surprisingly, in contrast to the excretion periods, the viral loads in stools did not increase in connection with age or immunosuppressive status. This fact may be one important piece in the pattern of high-level NoV transmissibility and may have an impact on the development of transmission prevention strategies.

Single-Point Mutations Causing More than 100-Fold Underestimation of Human Immunodeficiency Virus Type 1 (HIV-1) Load with the Cobas TaqMan HIV-1 Real-Time PCR Assay

ABSTRACT Systematic sequence analysis of human immunodeficiency virus type 1 (HIV-1) variants with RNA levels underestimated by the Cobas TaqMan HIV-1 assay demonstrated that mutations at a single position of the downstream primer can lead to the underestimation of HIV-1 RNA levels by more than 2 log and to false-negative results in minipool screening of blood donors. Mutations at this position are found in about 2% of all HIV-1 M gag sequences.

Transmission and disease association of Kaposi's sarcoma-associated herpesvirus: recent developments.

Purpose of review Kaposis sarcoma-associated herpesvirus or human herpesvirus 8, common in sub-Saharan Africa and around the Mediterranean Sea but rare in most other countries, is known to be transmitted in childhood within families in endemic regions, and through sexual contacts among high-risk groups in Western countries. Nevertheless recent developments on other modes of transmission of the virus have been made during the last years and are summarized in this review. Furthermore, recent published disease associations are discussed. Recent findings The last year has seen research addressing the question of parenteral transmission, sexual transmission through heterosexual contact, transmission of Kaposis sarcoma-associated herpesvirus-infected cells from organ donors to recipient, as well as the first suggestion that host genetic factors may facilitate infection in childhood. Additional clinical manifestations of infection with the virus such as primary pulmonary hypertension and germinotropic lymphoproliferative disorder have been identified. Summary Evidence of Kaposis sarcoma-associated herpesvirus transmission other than between homosexual adults and during childhood - namely transmission through heterosexual contact or injection drug use - is growing although these issues are still incompletely analysed and far away from being fully understood. Despite our increasing knowledge on transmission and disease associations of the virus, implications on the clinical management of associated diseases and public health have to be further evaluated in the coming years.

Kaposi's Sarcoma-Associated Herpesvirus Bacterial Artificial Chromosome Contains a Duplication of a Long Unique Region Fragment Within the Terminal Repeat Region

ABSTRACT Use of the Kaposis sarcoma-associated herpesvirus (KSHV) bacterial artificial chromosome 36 (KSHV-BAC36) genome permits reverse genetics approaches to study KSHV biology. While sequencing the complete KSHV-BAC36 genome, we noted a duplication of a 9-kb fragment of the long unique region in the terminal repeat region. This duplication covers a part of open reading frame (ORF) 19, the complete ORFs 18, 17, 16, K7, K6, and K5, and the putative ORF in the left origin of lytic replication, and it contains the BAC cassette. This observation needs to be kept in mind if viral genes located within the duplicated region are to be mutated in KSHV-BAC36.

Quantification of cytomegalovirus DNA levels in intestinal biopsies as a diagnostic tool for CMV intestinal disease

BACKGROUND CMV intestinal disease (CMV-ID) is a serious complication in immunocompromised patients and mainly diagnosed by clinical, endoscopic and histopathologic findings, whereas qualitative CMV-PCR in tissue samples is not recommended for diagnosis due to its low positive predictive value (PPV). OBJECTIVES To study the interpretation and diagnostic use of CMV-quantification by PCR in intestinal tissue biopsies to recognize CMV-ID. To develop cut-off intestinal CMV-loads attributing illness to CMV. STUDY DESIGN CMV-genome copies in 163 biopsies from the lower intestinal tract of immunocompromised patients were determined by quantitative real-time PCR, normalized to the cell number, and retrospectively compared to histopathological analysis, clinical findings and occurrence of CMV-antigenemia. Two cut-off intestinal CMV-loads, cut-off(histo) and cut-off(clin), were defined using histopathological or clinical criteria as gold standard, respectively. RESULTS CMV was detected in 32.5% of biopsies with a more than six log range of CMV-concentrations (1 x 10(-4)-1.4 x 10(2)copies/cell). Notably, biopsies with histopathologically or clinically confirmed CMV-ID had a significantly higher CMV-load (p<0.001). Cut-off(histo) and cut-off(clin) were defined at the intestinal CMV-load of 0.14 and 0.01 copies/cell, respectively, and improved the PPV. However, cut-off(histo) showed a decreased sensitivity for clinically defined CMV-ID cases. Interestingly, many patients with CMV-ID showed no concomitant CMV-antigenemia, suggesting a localized intestinal CMV-replication. CONCLUSIONS Quantification of CMV in intestinal biopsies is a useful diagnostic tool allowing the definition of cut-off values that can predict CMV-ID more accurate than qualitative PCR results. Further prospective studies have to clarify wether these cut-offs can improve diagnostics and treatment of CMV-ID in day-to-day clinical practice.

Symptomatic hepatitis B virus (HBV) reactivation despite reduced viral fitness is associated with HBV test and immune escape mutations in an HIV-coinfected patient.

Two sequential hepatitis B virus (HBV) strains obtained before and during an icteric flare-up of an occult HBV infection in a patient coinfected with human immunodeficiency virus revealed HBV surface antigen (HBsAg) test escape mutations, although the patient had never received hepatitis B-specific immunoglobulin. In contrast to the high HBV DNA loads, recurrence of HBsAg, and resulting icteric hepatitis, phenotypic analysis of the mutated HBV strains revealed significantly reduced replication efficacies in vitro, compared with wild-type HBV. Therefore, immune escape in the transiently anti-HBs-positive patient appeared to be crucial for persistence and reactivation. Immune escape mutants evolved even without exogenous selective pressure, hampered detection in HBsAg screening, and might be transmitted during reactivation with high HBV loads.

Next‐generation sequencing fails to identify human virus sequences in cutaneous squamous cell carcinoma

Nonmelanoma skin cancer (NMSC) shows a strongly increased incidence in solid organ transplant recipients (OTRs) and AIDS patients, suggesting an infectious etiology. The role of certain viruses, i.e., cutaneous human papillomaviruses (HPVs), in NMSC in immunosuppressed patients remains controversial. Merkel cell polyomavirus (MCPyV), which was recently identified using high‐throughput sequencing, has been linked to cutaneous proliferations. Here, we aimed to identify novel or known viral sequences at the transcript level in cutaneous squamous cell carcinomas (SCCs) from OTR by using 454 high‐throughput pyrosequencing, which can produce long reads (∼400 bp) and thus is better suited for the analysis of unknown sequences than other sequencing platforms. cDNA libraries from three OTR SCC biopsies were generated and submitted to next‐generation sequencing using a 454 platform. Bioinformatic analysis included digital transcriptome subtraction and—in parallel—reference mapping as an alternative way for depleting human sequences. All control sequences introduced for bioinformatics analysis were recovered correctly. Among 717,029 454‐sequenced transcripts, nearly all identified viral reads were derived from phages. Bacterial sequences originated from the skin flora or environmental sources. Our study did not reveal any transcripts of known oncogenic or related unknown human viruses. These findings suggest that there is no abundant expression of known human viruses, or viruses with a high degree of homology to known human viruses, in cutaneous SCCs of OTR. Further studies are required to exclude the presence of viruses in NMSC, which cannot easily be identified on the basis of sequence homology to known viruses.

CXCL13 as a Novel Marker for Diagnosis and Disease Monitoring in Pediatric PTLD

Posttransplant lymphoproliferative disease (PTLD) is a severe complication of immunosuppressive treatment in organ‐grafted children. Early diagnosis of PTLD is hampered by both unspecific clinical symptoms and lack of easy accessible markers. The homeostatic chemokine CXCL13, which plays a crucial role in B‐cell homing and lymphoid organ development, is expressed in some lymphomatous diseases. This study aims to investigate whether serum CXCL13 (sCXCL13) levels correlate with occurrence and regression of PTLD in pediatric solid‐organ graft recipients. Serum samples from PTLD patients (n = 21), patients with Epstein–Barr virus (EBV) reactivation (n = 18), and healthy age‐matched controls (n = 19) were tested for CXCL13 using a commercially available ELISA kit. sCXCL13 levels were significantly higher in PTLD patients than in healthy children. PTLD patients had also higher sCXCL13 values than pediatric solid‐organ recipients with EBV reactivation. An increase in sCXCL13 levels was observed from EBV reactivation to PTLD diagnosis in most cases. Elevated sCXCL13 levels were detected up to 2 years prior to PTLD diagnosis and correlated well with response to cytoreductive treatment in individual patients. sCXCL13, thus, may be a readily available surrogate marker for the diagnosis of PTLD and for monitoring of response to treatment in patients with initially elevated sCXCL13 levels.