Characteristics of Animal-assisted Interventions in the state of Lower Saxony, Germany, with a focus on hygiene in health care facilities.

Animal-assisted Interventions (AAIs) are becoming increasingly popular. To date, information on the extent of AAIs in Germany is limited. With a focus on infection control measures in health care facilities (HCFs), two studies were conducted in Lower Saxony to gain knowledge about the structure, characteristics and frequency of AAIs. An online survey among AAI providers identified dogs as the most important animal species in AAI, which mainly operated in educational facilities (53%) and/or on own property (46%). Twenty-nine percent offered their services in HCFs. The majority (55%) of the animal handlers was highly trained in AAI, but their awareness of hygiene and infection control measures to prevent zoonotic disease transmission was limited. Nineteen percent of animal handlers dewormed dogs only when faecal examinations were positive and 13% of dogs received ectoparasiticides only when infestations were present. Raw meat diets were frequent (82%). There was little awareness among animal handlers about the possibility of a zoonotic transmission from the client to the animal. Thus, handling of therapy dogs often reflected that of a "normal" pet ownership and did not always account for the special situation in HCFs. A telephone survey in 148 hospitals showed that 28% of the hospitals had experiences with animal-assisted therapies or animal visits, but 22% of these were lacking regulations on handling these animal contacts. While 28% of all hospitals had regulations for assistance dogs only 5% were aware of a new law that grants people accompanied by an assistance dog broad admission rights to public spaces, including HCFs. With an expected further increase in popularity of AAIs high quality standards which include infection control measures and animal welfare should be adopted by all AAI providers and recipients. This will ensure a safe implementation of this complementary medicine, where both sides - the human and the animal - can benefit.

EBV-specific T-cell immunity in pediatric solid organ graft recipients with posttransplantation lymphoproliferative disease.

BACKGROUND: Posttransplantation lymphoproliferative disease (PTLD) is an often Epstein-Barr virus (EBV)-associated mainly malignant complication after transplantation. We present data on EBV-specific T cells in children treated with rituximab with or without chemotherapy on the pediatric PTLD Pilot 2005 protocol. METHODS: Peripheral blood mononuclear cells were isolated from 16 pediatric patients with PTLD, 4 transplanted children with EBV reactivation, and 18 healthy controls. EBV-specific T cells were quantified by flow cytometric detection of intracellular interferon-γ after stimulation with autologous EBV-transformed lymphoblastoid cell lines and correlated with EBV load in peripheral blood. RESULTS: At diagnosis, PTLD patients had similar numbers of EBV-specific CD4 and CD8 T cells as healthy EBV-positive controls. EBV-specific T cells tended to be lower in early PTLD compared with late PTLD. During treatment with rituximab, CD4 and/or CD8 EBV-specific T cells increased in most patients, possibly reflecting restored immunocompetence due to a reduction of immunosuppression as well as antigenic stimulation by cross-presentation of EBV antigen from destroyed B cells. However, this increase did not predict response to rituximab or chemotherapy. EBV load and circulating B cells became undetectable in most patients during rituximab therapy. B-cell recovery after treatment was accompanied by redetection of EBV in peripheral blood, which was controlled by T-cell responses in 11 of 11 evaluable cases. CONCLUSIONS: In pediatric PTLD patients, pretreatment EBV-specific T-cell numbers are in the range of healthy controls. These cells increased on reduction of immunosuppression and treatment with rituximab. Recurrence of EBV viremia during complete remission is matched by strong T-cell responses.

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.

Kaposi's sarcoma-associated herpesvirus bacterial artificial chromosome contains a duplication of a long unique-region fragment within the terminal repeat region.

Use of the Kaposi's 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.

Comparison of the performance of direct fluorescent antibody staining, a point-of-care rapid antigen test and virus isolation with that of RT-PCR for the detection of novel 2009 influenza A (H1N1) virus in respiratory specimens.

Although infections with the novel pandemic 2009 influenza A (H1N1) virus (A/H1N1/2009) appeared to be relatively mild during the first summer of circulation ('off season'), there has been significant morbidity and hospitalization and several fatal cases. Thus, rapid detection of A/H1N1/2009 is crucial for efficient treatment and infection control measures. In contrast to seasonal influenza, where point-of-care (POC) rapid antigen tests and direct fluorescent antibody (DFA) staining ensure rapid detection, diagnosis of A/H1N1/2009 has so far been based on RT-PCR. This study retrospectively compared the performance of the Quidel QuickVue POC test, DFA staining and virus isolation with that of RT-PCR for A/H1N1/2009 detection in 526 respiratory specimens collected during the first wave of the outbreak from May to September 2009. A/H1N1/2009 was detected in 9.1% (48/526) of samples. One hundred and thirty-seven of the A/H1N1/2009 PCR-negative samples were additionally tested using a RealAccurate Respiratory RT-PCR panel, revealing other respiratory viruses (mainly entero/rhino- and adenoviruses) in 42.3% (58/137). All methods analysed detected A/H1N1/2009 with excellent specificity but different sensitivities (POC test: 18.2%; DFA staining: 38.7%; virus isolation: 45.7%). Therefore, the POC test was not suitable for diagnosis, detecting A/H1N1/2009 only if present in high concentrations (corresponding median Ct value=19.0; range=16.5-21.4). DFA staining was also able to detect A/H1N1/2009 in specimens with a lower virus concentration (median Ct value=24.0; range=16.5-29.8). Virus isolation, which was positive after a median time of 7.5 days, was too time-consuming. In summary, DFA staining is superior to POC testing and may be appropriate for patients expected to have a rather high level of virus replication. Nevertheless, in DFA-negative specimens, A/H1N1/2009 should be excluded by RT-PCR.

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.

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.

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.

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.

Rapid routine detection of enterovirus RNA in cerebrospinal fluid by a one-step real-time RT-PCR assay.

BACKGROUND AND OBJECTIVES: This study provides a one-step transcription/real-time (TaqMan probe) PCR assay (TM-PCR) with new consensus primer and probe sequences for generic detection of human pathogenic enteroviruses including difficult to detect ones like for instance Echovirus 30. The amplicon included parts of domain IV and V of the highly conserved internal ribosomal entry site. Generic detection was confirmed by testing a panel of 41 prototypes representing all five human enterovirus/poliovirus species. STUDY DESIGN AND RESULTS: The 95% detection limit was found to be 100 copies per run using in vitro transcribed coxsackievirus B3 RNA. TM-PCR was compared to an in house nested-PCR assay implemented in detecting enterovirus RNA from CSF samples of patients suffering from meningitis and encephalitis. Concordant results were obtained in all samples (11 positive, 101 negative). Specificity was confirmed with laboratory strains of other neurotropic viruses, and by testing 76 CSF samples of patients with encephalomyelitis disseminata, which all gave negative results. CONCLUSIONS: The new TM-PCR is a convincing alternative to conventional PCR protocols for the diagnosis of enterovirus meningitis. The one-step strategy limits hands on time and cross contamination risk combined with accelerated assay procedure of only 100 min.

Quantitative temporal and spatial distribution of adenovirus type 2 correlates with disease manifestations and organ failure during disseminated infection.

Disseminated adenovirus (HAdV) infections are serious complications in allogenic stem cell transplant (SCT) recipients. Quantitative HAdV DNA detection in blood samples demonstrated the association of high virus loads with disease and improved early diagnosis. However, the pathogenesis of disseminated HAdV disease, for example sources of HAdV DNA shedding in the blood stream and association of HAdV replication sites with disease manifestations, remained obscure. In this report, 24 bioptic and autoptic organ and tissue samples of an adult SCT recipient suffering from disseminated infection were quantitatively analyzed for HAdV DNA. Results indicate subsequent virus replication in the colon, bone marrow and liver as origin of HAdV DNAemia, which increased from 1.4 x 10(4) copies/ml to a peak of 2 x 10(9) copies/ml over a period of 84 days in spite of antiviral therapy. Symptoms as diarrhoea, bone marrow failure and hepatic failure were clearly linked to high HAdV DNA concentrations in affected organs. For example, the HAdV DNA level was 2.2 x 10(3) copies/cell in a colon biopsy when the patient suffered from diarrhoea whereas only 1.1 x 10(1) copies/cell were detected when symptoms had improved. Focal HAdV infection of the liver as demonstrated by laser microdissection was followed by fulminant virus replication with 1.3 x 10(5) copies of HAdV DNA/cell causing terminal hepatic failure. In conclusion, pathogenesis of disseminated HAdV disease was associated with virus replication in affected organs and not immune mediated as suggested recently by a fatal case of gene therapy with a non-replication competent HAdV-C5 vector.

Clinical relevance of and risk factors for HSV-related tracheobronchitis or pneumonia: results of an outbreak investigation.

INTRODUCTION: Herpes simplex virus (HSV) type 1 was identified in respiratory specimens from a cluster of eight patients on a surgical intensive care unit within 8 weeks. Six of these patients suffered from HSV-related tracheobronchitis and one from HSV-related pneumonia only. Our outbreak investigation aimed to determine the clinical relevance of and risk factors associated with HSV-related tracheobronchitis or pneumonia in critically ill patients, and to investigate whether the cluster was caused by nosocomial transmission. METHODS: A retrospective cohort study was performed to identify risk factors for the outcomes of HSV-related tracheobronchitis or pneumonia and death using univariable analysis as well as logistic regression analysis. Viruses were typed by molecular analysis of a fragment of the HSV type 1 glycoprotein G. RESULTS: The cohort of patients covering the outbreak period comprised 53 patients, including six patients with HSV-related tracheobronchitis and one patient with pneumonia only. HSV-related tracheobronchitis or pneumonia was associated with increased mortality (100% in patients with versus 17.8% in patients without HSV-related tracheobronchitis or pneumonia; P < 0.0001). The interaction of longer duration of ventilation and tracheotomy was associated with HSV-related tracheobronchitis or pneumonia in multivariable analysis. Identical HSV type 1 glycoprotein G sequences were found in three patients and in two patients. The group of three identical viral sequences belonged to a widely circulating strain. The two identical viral sequences were recovered from bronchoalveolar lavages of one patient with HSV-related tracheobronchitis and of one patient without clinical symptoms. These viral sequences showed unique polymorphisms, indicating probable nosocomial transmission. CONCLUSION: HSV-related tracheobronchitis or pneumonia is associated with increased mortality in critically ill patients. Care should be taken to avoid nosocomial transmission and early diagnosis should be attempted.

Risk of rabies infection and adverse effects of postexposure prophylaxis in healthcare workers and other patient contacts exposed to a rabies virus-infected lung transplant recipient.

BACKGROUND: Rabies virus was inadvertently transmitted to a lung transplant recipient through donor lungs. The patient was given ventilatory assistance and cared for postoperatively for 6 weeks before a diagnosis of rabies virus infection was made. Postexposure prophylaxis (PEP) was offered to potentially exposed healthcare workers (HCWs). METHODS: Only HCWs classified as belonging to possible and/or proven contact groups (according to a standardized interview) received PEP. The risk of individual HCWs being exposed to rabies virus was reassessed on the basis of viral concentrations measured in the patient's excretions and body fluids. HCWs who were vaccinated as part of PEP were followed up prospectively according to a standardized procedure. RESULTS: Of 179 HCWs and other patient contacts, 132 met the eligibility criteria for PEP (118 [89.4%] with possible contact and 14 [10.6%] with proven contact with the patient's excretions and/or body fluids). One hundred thirty-one individuals started PEP, and 126 met the inclusion criteria for analysis. Of these, 48 (38%) developed at least 1 adverse effect (8 [6.3%] had fever, 37 [29.4%] had headache, 3 [2.4%] had lymphadenopathy, 17 [13.5%] had dizziness, and 6 [4.8%] had paresthesia). No HCW or other patient contact developed rabies or serious PEP-related adverse effects. Reassessment of the individual's risk of infection as a function of the viral concentration in the patient's excretions and/or body fluids (up to 5.12 x 10(7) copies/mL) revealed that 103 HCWs (78.0%) had contact with high-risk substances (89 [67.40%] had possible contact and 14 [10.7%] had proven contact). CONCLUSION: HCWs can be exposed to significant viral concentrations in excretions and/or body fluids from rabies virus-infected lung transplant recipients. Because widespread use of PEP entails the possibility of significant health problems for HCWs considered to be at risk of contracting rabies, applying a rational indication for PEP is crucial.

Post-operative nosocomial infections after lung and heart transplantation.

BACKGROUND: Thoracic transplant recipients appear to be at high risk for post-operative infections. Therefore, we investigated the incidence and risk factors of post-operative nosocomial infections (NIs) in lung and heart transplant recipients. METHODS: From January 2002 to December 2003, a cohort of 208 consecutive thoracic transplant recipients (137 lung transplants [LTx], 51 heart transplants [HTx] and 20 combined transplants [CLTx]) were analyzed for post-operative infections and in-hospital mortality. NIs were determined according to CDC definitions. Uni- and multivariate risk factor analyses were performed. RESULTS: Of the 157 NIs, 59 were pneumonia (37.6%), 34 primary sepsis (21.6%), 34 urinary tract (21.6%) and 30 surgical site (19.1%). Despite a total NI incidence of 75.5%, more importantly 56.3% of all patients remained free from any infection. CLTx patients had a higher risk of developing NIs (odds ratio [OR] 4.97; 95% confidence interval [CI] 1.74 to 15.34). Risk factors for NIs were volume reduction procedures in LTx (OR 2.6; 95% CI 1.13 to 6.30) and re-do Tx (OR 5.25; 95% CI 1.41 to 26.8). In LTx patients, pre-operative colonization with gram-negative rods was found to be a risk factor for post-transplant pneumonia (OR 3.7; 95% CI 1.19 to 11.37). Presence of NI (OR 2.53; 95% CI 1.07 to 6.25) was a risk factor for mortality, as was cystic fibrosis (OR 3.20; 95% CI 1.27 to 7.92) and ventilation prior to transplantation (OR 4.00; 95% CI 1.28 to 12.09). CONCLUSION: The mortality risk associated with NIs requires close infection surveillance for developing specific preventive anti-infection strategies.

Absence of Kaposi's sarcoma-associated herpesvirus in patients with pulmonary arterial hypertension.

RATIONALE: In addition to Kaposi's sarcoma, Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) has been associated with two other diseases: primary effusion lymphoma and the plasma cell variant of multicentric Castleman's disease. Recently, evidence of KSHV infection was reported in plexiform lesions of idiopathic pulmonary arterial hypertension (IPAH) as well as in adjacent parenchyma and bronchial epithelial cells. OBJECTIVES: To further investigate a possible association of KSHV and pulmonary arterial hypertension. METHODS AND MEASUREMENTS: Twenty-six lungs explanted from German patients suffering from IPAH were tested for the presence of KSHV antigen and genomes by immunohistochemistry (IHC) and polymerase chain reaction (PCR). MAIN RESULTS: When stained with a commercial monoclonal antibody directed against the latency-associated nuclear antigen of KSHV, LANA-1, a positive signal reminiscent of the "speckled" nuclear pattern typical of latently KSHV-infected cells was found in 16 (61.5%) cases. Alveolar and bronchial epithelial cells in areas of unremarkable lung tissue, but not cells within the plexiform lesions, were the predominantly stained cell types. Different KSHV-PCR assays (based on orf26, orfK6, and orf72) performed on samples that had tested positively in IHC, however, could not confirm KSHV infection, indicating that the IHC signal was not due to KSHV infection. One IHC-negative patient tested positive by PCR. A PCR based on consensus degenerate hybrid oligonucleotide primers (CODEHOP) to detect yet unknown gamma-herpesviruses did not reveal any specific sequences. CONCLUSIONS: KSHV is unlikely to play a role in the pathogenesis of IPAH.

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

PURPOSE OF REVIEW: Kaposi's 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 Kaposi's 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 Kaposi's 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.

Quantitative multiplex real-time PCR for the sensitive detection of interferon beta gene induction and viral suppression of interferon beta expression.

Interferon-beta (IFN-beta) protein and activity can be detected by enzyme immunoassays and biological assays. However, precise quantification of low IFN-beta mRNA concentrations, which is advantageous for investigating IFN-beta gene expression in small tissue samples or during the early stage of a virus infection, remains a challenge. Therefore, we established a quantitative real-time PCR (qPCR) for IFN-beta and the housekeeping gene porphobilinogen deanimase (PBGD) in separated assays as well as in a multiplex procedure. Sensitivity for both the templates was less than 20 copies with an intra- and interassay variability of less than 5%. IFN-beta qPCR was utilized to optimize IFN-beta induction with dsRNA polyinosic-polycytidylic acid (poly I:C), delivered by a liposomal transfection agent for reproducible but low, non-cell-toxic IFN-beta concentrations. For studying coxsackievirus B3 (CVB3) interference with IFN-beta expression, CVB3 infected fibroblasts were induced with poly I:C. A significant reduction of IFN-beta mRNA but not PBGD mRNA was demonstrated 5 h after CVB3 infection, indicating a specific inhibition of IFN-beta expression by CVB3 on the mRNA level, in addition to previously reported effects on the translation/post-translation level. In conclusion, sensitive IFN-beta/PBGD multiplex qPCR proved to be a useful tool to study viral interaction with IFN-beta expression.

Activation of mitogen-activated protein kinase and NF-kappaB pathways by a Kaposi's sarcoma-associated herpesvirus K15 membrane protein.

The K15 gene of Kaposi's 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 gamma(2)-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-kappaB pathways, as well as (more weakly) the c-Jun N-terminal kinase/SAPK pathway. Activation of the MAPK and NF-kappaB 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.