Clonal deleted latent membrane protein 1 variants of Epstein-Barr virus are predominant in European extranodal NK/T lymphomas and disappear during successful treatment.

Extranodal natural killer/T-cell lymphomas (NK/TL), rare in Europe, are Epstein-Barr virus (EBV) associated lymphomas with poor outcomes. Here, we determined the virus type and analyzed the EBV latent membrane protein-1 (LMP1) gene sequence in NK/TL from French patients. Six clones of viral LMP1 were sequenced by Sanger technology in blood from 13 patients before treatment with an l-asparaginase based regimen and, for 8 of them, throughout the treatment. Blood LMP1 sequences from 21 patients without any known malignancy were tested as controls. EBV Type A was identified for 11/13 patients and for all controls. Before treatment, a clonal LMP1 gene containing a 30 bp deletion (del30) was found in 46.1% of NK/TL and only in 4.8% of controls. Treatment was less effective in these patients who died more rapidly than the others. Patients with a deleted strain evolving toward a wild-type strain during treatment reached complete remission. The LMP1 gene was sequenced by highly sensitive next-generation sequencing technology in five NK/TL nasopharyngeal biopsies, two of them originating from the previous patients. Del30 was present in 100% of the biopsies; two viruses at least coexisted in three biopsies. These results suggest that del30 may be associated with poor prognosis NK/TL and that strain evolution could be used as a potential marker to monitor treatment.

Epstein-Barr Virus (EBV) Is Mostly Latent and Clonal in Angioimmunoblastic T Cell Lymphoma (AITL).

The Epstein-Barr virus (EBV) is associated with angioimmunoblastic T cell lymphoma (AITL), a peripheral T lymphoma of poor prognosis in at least 90% of cases. The role of EBV in this pathology is unknown. Using next-generation sequencing, we sequenced the entire EBV genome in biopsies from 18 patients with AITL, 16 patients with another EBV-associated lymphoma, and 2 controls. We chose an EBV target capture method, given the high specificity of this technique, followed by a second capture to increase sensitivity. We identified two main viral strains in AITL, one of them associated with the mutations BNRF1 S542N and BZLF1 A206S and with mutations in the EBNA-3 and LMP-2 genes. This strain was characterized in patients with short post-diagnosis survival. The main mutations found during AITL on the most mutated latency or tegument genes were identified and discussed. We showed that the virus was clonal in all the AITL samples, suggesting that it may be involved in this pathology. Additionally, EBV was latent in all the AITL samples; for one sample only, the virus was found to be latent and probably replicative, depending on the cells. These various elements support the role of EBV in AITL.

Comprehensive Epstein-Barr Virus Transcriptome by RNA-Sequencing in Angioimmunoblastic T Cell Lymphoma (AITL) and Other Lymphomas.

The Epstein-Barr virus (EBV) is associated with angioimmunoblastic T cell lymphoma (AITL) in more than 80% of cases. Few studies have focused on this association and it is not clear now what role the virus plays in this pathology. We used next-generation sequencing (NGS) to study EBV transcriptome in 14 AITLs compared to 21 other lymphoma samples and 11 cell lines including 4 lymphoblastoid cell lines (LCLs). Viral transcripts were recovered using capture probes and sequencing was performed on Illumina. Bam-HI A rightward transcripts (BARTs) were the most latency transcripts expressed in AITLs, suggesting they may play a role in this pathology. Thus, BARTs, already described as highly expressed in carcinoma cells, are also very present in AITLs and other lymphomas. They were poorly expressed in cell lines other than LCLs. AITLs showed a latency IIc, with BNLF2a gene expression. For most AITLs, BCRF1, which encodes a homologous protein of human interleukin 10, vIL-10, was in addition expressed. This co-expression can contribute to immune escape and survival of infected cells. Considering these results, it can be assumed that EBV plays a pathogenic role in AITLs.

Amoxicillin-induced flare in patients with DRESS (Drug Reaction with Eosinophilia and Systemic Symptoms): report of seven cases and demonstration of a direct effect of amoxicillin on Human Herpesvirus 6 replication in vitro.

Amoxicillin is known to induce exanthema in patients with EBV-induced infectious mononucleosis. It is widely recognized that the reactivation of herpesviruses, including HHV-6 (Human Herpesvirus 6) and EBV (Epstein Barr virus) is associated with DRESS (Drug Reaction with Eosinophilia and Systemic Symptoms). We report 7 cases of amoxicillin-induced flare in patients with DRESS induced by other drugs and investigate whether amoxicillin may have a direct effect on HHV-6 replication in vitro. 7 cases of DRESS with amoxicillin-induced flare were retrospectively analysed. The influence of amoxicillin on HHV-6 HST strain replication was studied in vitro in a human T lymphoblastoid MT4 cell line. The viral replication was quantified by immunofluorescence assay and by real-time polymerase chain reaction (PCR). Comparisons were performed using the Student's t test. Amoxicillin-induced flare was observed in 7 patients with DRESS induced by other drugs. In two cases HHV-6 reactivation was studied and was demonstrated by PCR. Amoxicillin neither modified cell viability nor cell proliferation for the range of tested concentrations. Amoxicillin increased the replication of HHV-6 at 25 microg*mL-1 and 50 microg*mL-1. Amoxicillin may induce a flare of DRESS, possibly by acting directly on herpesvirus replication.

The survival of herpes simplex virus in preserved murine corneas.

BACKGROUND/AIMS: This study investigates the survival of HSV in infected mouse corneas, in the conditions of normal human eye bank preservation. METHODS: Hundred seventy-two BALB/C mice infected with herpes simplex virus 1 (HSV-1) (KOS) were randomly assigned to either: no preservation in group 1 (n = 62), 31 degrees C preservation for 3 weeks in group 2 (n = 70) or 4 degrees C preservation for 8 days in group 3 (n = 40). The presence of HSV-1 was thereafter detected by viral culture and PCR. RESULTS: In groups 1, 2 and 3, HSV-1 was detected by culture in 22 (35.5%), 1 (1.4%) and 0 (0.0%) of the corneas, and by PCR in 27 (43.7%), 3 (4.2%) and 7 (17.5%) of the corneas respectively. When compared to group 1, HSV was detected significantly less often in groups 2 (p < 0.0001) and 3 (p < 0.0001). CONCLUSIONS: HSV-1 DNA undergoes a degradation during corneal preservation.

HIV-2 Primary Infection in a French 69-Year-Old Bisexual Man.

More than 1 million individuals, mainly in West Africa, are thought to be infected with HIV-2. Acute HIV-2 infection is rarely observed, only 2 primary infections have been described to date. We report a detailed case of HIV-2 primary infection in a 69-year-old French bisexual Caucasian man, thereby providing valuable insights into HIV-2 early infection.

Multiplex real-time PCR assay for simultaneous quantitation of human cytomegalovirus and herpesvirus-6 in polymorphonuclear and mononuclear cells of transplant recipients.

Human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6) are two closely related viruses, which belong to the Herpesviridae family. Following primary infection, they are thought to persist for life as latent forms in mononuclear cells. HCMV and HHV-6 can cause considerable morbidity in immunocompromised individuals, such as transplant patients. A sensitive and specific LightCycler multiplex real-time PCR assay based on fluorescence energy transfer (known as FRET) was developed. This assay, by using two sets of hybridization probes specific for HHV-6 (A and B) and HCMV, can differentiate reliably and quantify simultaneously both viruses in order to diagnose reactivation processes. The assay was optimized and the lower limit of detection for both viruses was determined to be 10 viral genome copies per reaction. Both viruses were quantified in 83 peripheral blood mononuclear cells (PBMCs) and 87 polymorphonuclear leukocytes (PMNLs) collected from 32 transplant recipients. This multiplex real-time quantitative PCR was finally compared with two other quantitation and detection assays used daily in laboratory (PCR DIG detection and antigenemia for HCMV, TaqMan Assay for HHV-6). This technique can be useful for the differentiation and quantitation of HCMV and HHV-6 for monitoring transplant patients.

A reverse transcription-nested PCR assay for HHV-6 mRNA early transcript detection after transplantation.

Monitoring of human herpesvirus-6 (HHV-6) reactivation is important, especially in immunocompromised patients such as transplant recipients. Reverse transcription PCR (RT-PCR) is a useful method to distinguish between latent and active infection. Here, a RT-nested PCR coupled with a colorimetric plate hybridization assay was established to detect HHV-6 types A and B U79/80 mRNAs. After confirming the reliability of the assay on HHV-6 cultures, it was applied to the detection of HHV-6 reactivation after renal (27 patients), bone marrow (14 patients) or heart (7 patients) transplantation. A total of 206 blood samples were tested from renal (137), bone marrow (58) and heart (11) transplant recipients. U79/80 mRNAs were found in 32 samples that were considered as indicative of HHV-6 reactivation: 15, 13 and 5 from kidney, bone marrow and heart transplant recipients, respectively. Finally, U79/80 mRNA detection was correlated with clinical manifestations including leucopenia, skin rash, graft rejection or dysfunction and diarrhoea.

Transmission of viruses through corneal transplantation.

Transmission of viruses through transplantation has become a major concern for surgeons and tissue banks. Keratoplasty has been the first transplantation procedure in which transmission of a virus from the donor to the recipient has been proven, and recently, herpes has been suggested as a transmittable disease via corneal transplantation. In this article we review the history of viral transmission through transplantation, especially focusing on keratoplasty, and develop the recent updates on transmission of HSV through corneal grafting, its implications for surgeons and eye banks, and further insights into viral security in eye banks.

Hepatitis B mother--to--child transmission.

Approximately 350 million people are estimated to be chronically infected with hepatitis B virus, leading to an important public health problem. In highly endemic areas where 8 to 15% of people are chronically infected with hepatitis B virus, the risk for the neonate to be perinatally infected by the chronically infected mother, then to become chronically infected themselves, is very high. In those countries, the World Health Organization recommends hepatitis B vaccination systematically at birth, independent of hepatitis B virus maternal status. This vaccination program has begun to induce a rapid decrease in the number of acute hepatitis B virus infections and has also had a secondary effect of a decrease in related sequels. Lamivudine (Zeffix, GlaxoSmithKline), when associated with the immunization of the neonate, was recently demonstrated to dramatically reduce the residual risk of perinatal transmission. In intermediate and low endemicity areas, a systematic hepatitis B surface antigen screening is recommended during pregnancy, allowing, in the case of positivity, a selective hepatitis B virus neonate immunization during the first 12 h of life. Hepatitis B virus vaccination of children born to hepatitis B surface antigen-positive mothers confers long-term immunity.

DRESS syndrome.

Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome, initially recognized as a serious form of cutaneous drug adverse reaction, is now viewed as a drug-related syndrome that can cause life-threatening organ dysfunctions. Characteristic features include a long time interval from first drug exposure to symptom onset and a prolonged course, often with flares, even after discontinuation of the causal drug. The pathophysiology of DRESS syndrome remains incompletely understood but involves reactivation of herpes viruses (HHV-6, HHV-7, EBV, and CMV), against which the body mounts a strong immune response. The culprit drugs may not only affect epigenetic control mechanisms, thereby promoting viral reactivation, but also induce an antiviral T-cell response by interacting with the major histocompatibility complex receptor in individuals with genetic susceptibility factors. Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) syndrome is a potentially life-threatening form of cutaneous drug adverse reaction. The severity of this syndrome is related to the systemic manifestations, which can result in multiorgan failure. DRESS syndrome is characterized by highly specific features, most notably regarding the timing of the manifestations. New insights into the underlying pathophysiological mechanisms indicate a role for immunogenetic susceptibility factors and for reactivation of human herpes viruses (HHVs), chiefly HHV-6. We report a typical case of DRESS syndrome and discuss recent data about this condition.

Saliva polymerase chain reaction assay for detection and follow-up of herpesvirus reactivation in patients with drug reaction with eosinophilia and systemic symptoms (DRESS).

IMPORTANCE: Reactivations of human herpesviruses (HHVs) contribute to the development of drug reaction with eosinophilia and systemic symptoms (DRESS). Diagnosis of HHV reactivation is conventionally based on quantitative real-time polymerase chain reaction (PCR) analysis of blood samples, but these viruses are present in the oropharynx and are shed in saliva. OBJECTIVE: To evaluate the use of a saliva PCR assay for demonstrating HHV shedding in patients with DRESS. DESIGN: Shedding of HHV in saliva was prospectively studied in patients with DRESS. Reactivations of HHV, including HHV-6, HHV-7, cytomegalovirus (CMV), and Epstein-Barr virus (EBV), were studied by performing quantitative real-time PCR analysis of blood samples obtained at admission) and serial samples of saliva obtained within the first 2 weeks of DRESS; saliva samples from controls were compared. PARTICIPANTS: The study included 5 patients who met definite criteria for DRESS and 15 controls (5 immunosuppressed patients and 10 healthy adults). MAIN OUTCOME MEASURES: DNA viral loads of HHV, including HHV-6, HHV-7, CMV, and EBV as measured with real-time PCR in blood and saliva samples from patients with DRESS and saliva samples from immunosuppressed and healthy controls. RESULTS: The PCR assay demonstrated shedding of HHV-7, EBV, HHV-6, and CMV, listed by order of magnitude. The DNA viral loads in blood and saliva samples, also measured with real-time PCR, were found to be close. In 1 patient, reactivations in saliva preceded clinical manifestations of CMV disease. Significant production of HHV-7 and EBV was demonstrated in saliva samples from the controls, but neither HHV-6 nor CMV were detected. CONCLUSIONS AND RELEVANCE: The saliva PCR assay is a useful tool for demonstration and follow-up of HHV reactivation. The interpretation of HHV viral loads in saliva differs according to HHV type.

Intrathecal synthesis of specific antibodies as a marker of herpes simplex encephalitis in patients with negative PCR.

BACKGROUND: PCR in the cerebrospinal fluid (CSF) has become the sole method used for the diagnosis of herpes simplex encephalitis (HSE). Nevertheless, PCR results may sometimes be false negative, and in this situation other techniques may be useful. METHODS: 3 patients hospitalised for meningoencephalitis with fever showed a negative result for herpes simplex virus (HSV) PCR in their CSF. We then performed a detection of intrathecal anti-HSV immunoglobulins (IgGs) in the CSF and analysed their level in relation to those in the serum, compared to albumin. RESULTS: We confirmed that IgG synthesis was the direct consequence of an immune system reaction in the 3 patients' CSF. These results were consistent with clinical signs and neurodiagnostic procedures. They prompted us to continue the treatment, which would have been stopped following the negative PCR results. The clinical progression was favourable for all patients. CONCLUSIONS: PCR, which many physicians now consider the gold standard for the detection of HSV, may sometimes yield false negative results, i.e. when performed too early after the disease onset or when the viral load is too low. The method described here, although positive a few days after PCR, may prove helpful in the diagnosis of HSE for patients with negative HSV PCR in the CSF.

Involvement of human herpesvirus-6 variant B in classic Hodgkin's lymphoma via DR7 oncoprotein.

PURPOSE: Hodgkin's lymphoma (HL) is associated with the presence of EBV in Reed-Sternberg (RS) cells in ∼40% of cases. Here, we studied the presence of human herpesvirus type 6 (HHV-6) variant B in RS cells of HL patients and correlated results with clinical parameters. We then examined the implication of HHV-6 DR7B protein in cell deregulation. EXPERIMENTAL DESIGN: HHV-6 DR7B protein was produced in a Semliki Forest virus system. Polyclonal antibodies were then generated and used for immunochemical HHV-6 localization in HL biopsies. Binding between DR7B and p53 was studied using a double-hybrid system. Transactivation of NFκB was observed after transient transfection using reporter gene assays. We looked for Id2 factor expression after stable transfection of the BJAB cell line by reverse transcription-PCR and Western blot analysis. RESULTS: HHV-6 was more common in nodular sclerosis subtype HL, and DR7B oncoprotein was detected in RS cells for 73.7% of EBV-negative patients. Colocalization of EBV and HHV-6 was observed in RS cells of doubly infected patients. DR7B protein bound to human p53 protein. p105-p50/p65 mRNA expression and activation of the NFκB complex were increased when DR7B was expressed. Stable expression of DR7B exhibited a strong and uniform expression of Id2. A slightly higher percentage of remission was observed in patients with RS cells testing positive for DR7B than in those testing negative. CONCLUSIONS: Collectively, these data provide evidence for the implication of a novel agent, HHV-6, in cases of nodular sclerosis HL.

Twelve-year analysis of severe cases of drug reaction with eosinophilia and systemic symptoms: a cause of unpredictable multiorgan failure.

BACKGROUND: Factors implicated in the severity of drug reaction with eosinophilia and systemic symptoms (DRESS) have not been identified. We retrospectively describe and analyze severe cases of DRESS defined by history of intensive care unit admission and death due to DRESS. OBSERVATIONS: Of 15 patients retrospectively recruited in France, 14 were admitted to the intensive care unit and 3 died. The culprit drugs were already known to cause or trigger DRESS: allopurinol, minocycline hydrochloride, anticonvulsants, sulfonamides, and antibiotics. Visceral involvement with severe manifestations responsible for intensive care unit admission or death was variable and often multiple (pneumonitis, hepatitis, renal failure, encephalitis, hemophagocytosis, cardiac failure, and pancytopenia) and resulted in multiorgan failure in 11 patients. These severe complications sometimes developed late in DRESS. Human herpesvirus 6 infection was demonstrated in 6 of 7 patients. In addition, human herpesvirus 6 infection was demonstrated in involved viscera in 2 patients. CONCLUSIONS: Severe DRESS is rare. Some specificities of visceral involvement were associated with allopurinol and minocycline. However, visceral involvement comprising multiorgan failure seemed to be unpredictable. Better knowledge of DRESS is necessary to propose specific and prompt treatment. Early demonstration of human herpesvirus 6 reactivation could be considered a prognostic factor for identifying patients at higher risk and, hence, needs to be evaluated.

HHV-6 and EBV DNA quantitation in lymph nodes of 86 patients with Hodgkin's lymphoma.

Human herpesvirus (HHV-6) and Epstein-Barr virus (EBV), are two ubiquitous human herpesviruses which share many common features although they belong to different sub-families. In particular, both viruses are found in lymph nodes of patients suffering from Hodgkin's lymphoma. The aim of this study was to detect and to quantify independently HHV-6 and EBV by a real-time PCR in lymph nodes from 86 patients with Hodgkin's lymphoma. EBV quantitative method was compared with LMP-1 protein detection among the same samples. EBV genome was detected for 61.6% of the patients (53/86) and the highest prevalence of this virus was observed in Hodgkin's lymphoma with mixed-cellularity histopathological type (80%). In contrast to that, HHV-6 genome was detected for 79.1% of the patients (68/86) and was most observed in the nodular-sclerosis group (83.6%). Among the 68 HHV-6 positive samples, 63 belonged to the B subtype. A large number of biopsies (47.7%) were positive for both viruses whereas a little number (7%) was negative for both. EBV quantitation and LMP-1 immunohistochemistry were correlated statistically but this latter technique was less sensitive. Among the nodular-sclerosis patients, HHV-6-/EBV+ patients were significatively older than HHV-6+/EBV- patients. Patients infected dually had higher values of quantitation for each virus than those positive for one virus. Data of the clinical follow-up obtained by diagnosis and during the treatment of 83 patients, were correlated with the virological findings.

Specific antibody production in herpes keratitis: intraocular inflammation and corneal neovascularisation as predicting factors.

PURPOSE: The purpose of the study is to investigate whether analysis of specific antibody synthesis can aid the diagnosis of herpes keratitis. METHODS: Aqueous humor was collected from 39 patients with presumed recurrent herpes keratitis, including 23 consulting for keratitis and 16 patients scheduled for penetrating keratoplasty. Local antibody production was ascertained by analysis of paired aqueous humor/serum samples, using a modified micro-ELISA technique. RESULTS: Local production of antibodies was found in 32 patients (82%): anti-herpes simplex virus (HSV) antibodies in 26 (67%) and anti-varicella zoster virus (VZV) antibodies in 11 (28%). Twenty of 23 patients with active keratitis (87%), and 12 of 16 undergoing keratoplasty (75%), tested positive. Five patients had local production of both anti-HSV and anti-VZV antibodies, whereas seven patients tested negative. Local antibody production was significantly associated with intraocular inflammation (P<0.05), corneal neovascularisation (P<0.05), and positive response to anti-viral treatment (P<0.05). No complications were encountered in sampling aqueous humor. CONCLUSIONS: Assessment of local anti-HSV and -VZV antibody production is a safe and reliable diagnostic procedure for recurrent herpes keratitis. It might be particularly helpful in patients presenting with intraocular inflammation and neovascularisation since it discriminates between herpes and non-herpes pathologies and may therefore be useful for preventive and therapeutic strategies.

Main adult herpes virus infections of the CNS.

Herpes viruses are widely involved in human infectious diseases, and some are life threatening, such as CNS infections. These manifestations vary according to the type of virus involved and the immune status of the patient. This article will review the clinical manifestations (encephalitis, myelitis, meningitis and postinfectious encephalomyelitis), the diagnostic strategies and the presently used drugs (acyclovir, valacyclovir, ganciclovir, valgancyclovir, foscarnet and cidofovir). The review will also discuss drugs that are currently in the pipeline and that could be used in the future.

Successful treatment of human herpesvirus 6 encephalomyelitis in immunocompetent patient.

We report a case of human herpesvirus 6 (HHV-6) encephalomyelitis in an immunocompetent patient, which was confirmed by viral amplification from cerebrospinal fluid. Cidofovir was used, followed by ganciclovir, because of an adverse effect to probenecid. The patient recovered. HHV-6 should be recognized as one of the causes of encephalomyelitis.