Background Contemporary descriptions of classical Hodgkin lymphoma (cHL) are lacking from sub-Saharan Africa where human immunodeficiency virus (HIV) and Epstein–Barr virus (EBV) are prevalent. Methods We describe a prospective cHL cohort in Malawi enrolled from 2013 to 2015. Patients received standardized treatment and evaluation, including HIV status and EBV testing of tumors and plasma. Results Among 31 patients with confirmed cHL, the median age was 19 years (range, 2–51 years) and 22 (71%) were male. Sixteen patients (52%) had stage III/IV, 25 (81%) B symptoms, and 16 (52%) performance status impairment. Twenty-three patients (74%) had symptoms >6 months, and 11 of 29 (38%) had received empiric antituberculosis treatment. Anemia was common with median hemoglobin 8.2 g/dL (range, 3.1–17.1 g/dL), which improved during treatment. No children and 5 of 15 adults (33%)were HIV+. All HIV+ patients were on antiretroviral therapy for a median 15 months (range, 2–137 months), with median CD4 count 138 cells/μL (range, 23–329 cells/μL) and four (80%) having undetectable HIV. EBV was present in 18 of 24 (75%) tumor specimens, including 14 of 20 (70%) HIV− and 4 of 4 (100%) HIV+. Baseline plasma EBV DNA was detected in 25 of 28 (89%) patients, with median viral load 4.7 (range, 2.0–6.7) log10copies/mL, and subsequently declined in most patients. At 12 months, overall survival was 75% (95% confidence interval [CI], 55%–88%) and progression-free survival 65% (95% CI, 42%–81%). Baseline plasma EBV DNA and persistent viremia during treatment were associated with poorer outcomes. Conclusion cHL in Malawi is characterized by delayed diagnosis and advanced disease. Most cases were EBV associated and one-third of adults were HIV+. Despite resource limitations, 12-month outcomes were good.

Point-of-care tools are needed in sub-Saharan Africa (SSA) to improve pediatric Burkitt lymphoma (BL) diagnosis and treatment. We evaluated plasma Epstein-Barr virus (pEBV) DNA as a pediatric BL biomarker in Malawi. Prospectively enrolled children with BL were compared to classical Hodgkin lymphoma (cHL) and non-lymphoma diagnoses. Pediatric BL patients received standardized chemotherapy and supportive care. pEBV DNA was measured at baseline, mid-treatment, and treatment completion. Of 121 assessed children, pEBV DNA was detected in 76/88 (86%) with BL, 16/17 (94%) with cHL, and 2/16 (12%) with non-lymphoma, with proportions higher in BL versus non-lymphoma (p<0.001) and similar in BL versus cHL (p=0.69). If detected, median pEBV DNA was 6.1 log10copies/mL for BL, 4.8 log10copies/mL for cHL, and 3.4 log10copies/mL for non-lymphoma, with higher levels in BL versus cHL (p=0.029), and a trend toward higher levels in BL versus non-lymphoma (p=0.062). pEBV DNA declined during treatment in the cohort overall and increased in several children before clinical relapse. Twelve-month overall survival was 40% in the cohort overall, and for children with baseline pEBV detected, survival was worse if baseline pEBV DNA was ≥6 log10copies/mL versus <6 log10copies/mL (p=0.0002), and also if pEBV DNA was persistently detectable at mid-treatment versus undetectable (p=0.041). Among children with baseline pEBV DNA detected, viremia was the only significant risk factor for death by 12 months in multivariate analyses (adjusted hazard ratio 1.35 per log10copies/mL, 95% CI 1.04–1.75, p=0.023). Quantitative pEBV DNA has potential utility for diagnosis, prognosis, and response assessment for pediatric BL in SSA.

Summary Kaposi’s sarcoma-associated herpesvirus (KSHV) is linked to human malignancies. The majority of tumor cells harbor latent virus and a small percentage undergo spontaneous lytic replication. Both latency and lytic replication are important for viral pathogenesis and spread but the cellular players involved in the switch between the two viral lifecycle phases are not clearly understood. We conducted a siRNA screen targeting the cellular kinome and identified Tousled-like kinases (TLKs) as cellular kinases that control KSHV reactivation from latency. Upon treatment of latent KSHV-infected cells with siRNAs targeting TLKs, we saw robust viral reactivation. Knockdown of TLKs in latent KSHV-infected cells induced expression of viral lytic proteins and production of infectious virus. TLKs were also found to play a role in regulating reactivation from latency of another related oncogenic gammaherpesvirus, Epstein-Barr virus (EBV). Our results establish the TLKs as cellular repressors of gammaherpesviral reactivation.

The K1 gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) is encoded by the first open reading frame (ORF) of the viral genome. To investigate the role of the K1 gene during the KSHV life cycle, we constructed a set of recombinant viruses that contained either wild-type (WT) K1, a deleted K1 ORF (KSHV⌬K1), stop codons within the K1 ORF (KSHV-K1 5؋STOP ), or a revertant K1 virus (KSHV-K1 REV ). We report that the recombinant viruses KSHV⌬K1 and KSHV-K1 5؋STOP displayed significantly reduced lytic replication compared to WT KSHV and KSHV-K1 REV upon reactivation from latency. Additionally, cells infected with the recombinant viruses KSHV⌬K1 and KSHV-K1 5؋STOP also yielded smaller amounts of infectious progeny upon reactivation than did WT KSHV-and KSHV-K1 REV -infected cells. Upon reactivation from latency, WT KSHV-and KSHV-K1 REV -infected cells displayed activated Akt kinase, as evidenced by its phosphorylation, while cells infected with viruses deleted for K1 showed reduced phosphorylation and activation of Akt kinase. Overall, our results suggest that K1 plays an important role during the KSHV life cycle. IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of three human malignancies, and KSHV K1 is a signaling protein that has been shown to be involved in cellular transformation and to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. In order to investigate the role of the K1 protein in the life cycle of KSHV, we constructed recombinant viruses that were deficient for K1. We found that K1 deletion viruses displayed reduced lytic replication compared to the WT virus and also yielded smaller numbers of infectious progeny. We report that K1 plays an important role in the life cycle of KSHV. K aposi's sarcoma (KS)-associated herpesvirus (KSHV), also known as human herpesvirus 8, is the causative agent of KS, a vascular neoplasm of endothelial cell origin (1). KSHV infection is linked to two B cell lymphoproliferative disorders: primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman's disease (MCD) (2-4). KSHV predominantly displays a latent state in infected cells and in KSHV-associated tumors, but a small percentage of KSHV-infected cells undergo reactivation, which is thought to be important for KS tumorigenesis (5). KSHV reactivation can occur through multiple events (6, 7), and the KSHV replication and transcription activator (RTA) protein is the only viral protein that is both necessary and sufficient to reactivate KSHV. The expression of RTA leads to the activation of downstream lytic genes and ultimately the production of progeny virions (8-12).K1 is a transmembrane glycoprotein encoded by the first open reading frame (ORF) in the KSHV genome (13). Although K1 is highly upregulated during the lytic cycle, it has also been shown to be expressed at lower levels during latency (14). K1 is expressed in KS lesions and primary effusion lymphoma cell lines (13-17). The K1 protein contains an immunoreceptor tyrosi...

A lthough a number of antiviral drugs are effective inhibitors of Epstein-Barr virus (EBV) replication and are used empirically, none is of proven effectiveness for treatment of EBV infection (1, 2). Maribavir (MBV), which is in late-stage clinical trials for use against human cytomegalovirus (HCMV) infection in allogeneic stem cell and bone-marrow transplant recipients (3,4), is of special interest because it is also a potent inhibitor of EBV replication (5-7). In stem-cell and organ transplant recipients, EBV infection poses the hazard of generating B-cell lymphomas that are ultimately fatal. While no drugs are currently approved for treatment of EBV disease, several that inhibit EBV are available, and these can be divided into two main classes: those that target the viral DNA polymerase and those that function independently of it (8-12). Acyclic nucleoside and phosphonated nucleotide analogs, as well as pyrophosphate analogs, all target the viral polymerase.A new class of HCMV inhibitors, benzimidazole compounds, with more specific antiviral properties and fewer adverse side effects, blocked HCMV DNA maturation and encapsidation processes and led to the design of 1-H-␤-L-ribofuranoside-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir [MBV]) (13)(14)(15)(16)(17)(18)(19)(20). Unlike its parent compound, which inhibits HCMV replication but not EBV replication, MBV inhibits both (7,21). Inhibitory effects of MBV are produced mainly through inhibition of the HCMV and EBV protein kinases (PK) (21-24). Previous phase 3 studies with a dosage of 100 mg twice a day (BID) did not have sufficient activity to prevent HCMV disease, but the safety profile and data from case studies suggested that higher doses would be clinically active (3). MBV is now in new phase 2 trials at doses of 400, 800, and 1,200 mg BID (3).Maribavir selectively inhibits the HCMV protein kinase, UL97, determined by direct inhibition of kinase activity in vitro and by genetic mapping of the MBV-resistant phenotype (21). MBV also inhibits the EBV protein kinase (BGLF4), resulting in inhibition of phosphorylation of the EBV DNA processivity factor BMRF1, but does not seem to act directly on the EBV kinase in vitro (7,24).We have recently found that MBV also inhibits expression of multiple EBV transcripts, in contrast to acyclovir (ACV), which has little effect on EBV RNAs. Thus, MBV has a unique dual effect on viral DNA transcription as well as replication (25). In this study, we find that the inhibitory profile of MBV transcripts is similar to that produced by mutant EBV in which PK expression and activity have been knocked out (26). Thus, the results suggest that MBV largely affects EBV transcript levels through inhibition of BGLF4.To determine if the profile of viral transcripts produced by MBV is mediated by the viral kinase, we utilized BGLF4 knockout (KO) (dBGLF4/NeoST) and revertant (dBGLF4/NeoSt/R) viruses constructed and characterized by Murata et al. (26). 293 cells maintaining wild-type (WT), BGLF4 knockout, and revertant EBV genomes (2...