Berthe-Marie Imbert-Marcille

Human Herpesvirus (HHV)-6 and HHV-7: Two Closely Related Viruses with Different Infection Profiles in Stem Cell Transplantation Recipients

Human herpesvirus (HHV)-6 and HHV-7 loads were evaluated retrospectively in peripheral blood mononuclear cells (PBMC) from 78 recipients of stem cell transplantation (SCT) by real-time polymerase chain reaction. The median HHV-6 load in patients was 1357 genome equivalent copies (EqCop)/10(6) PBMC but was below the quantitation threshold in 31 immunocompetent individuals, which strongly suggests that HHV-6 reactivation occurred after SCT. The HHV-6 load was higher in patients with delayed neutrophil engraftment (P=.002) or severe graft-versus-host disease (P=.009). Moreover, the occurrence of at least 1 HHV-6-related manifestation (fever, cutaneous rash, pneumonitis, or partial myelosuppression) was statistically associated with a concomitant virus load >10(3) EqCop/10(6) PBMC (P=.007). Conversely, HHV-7 reactivation was not favored, because median HHV-7 loads were similar in patients and healthy control subjects (1053 vs. 1216 EqCop/10(6) PBMC). The kinetics of Roseolovirus loads during the posttransplantation period suggested that HHV-7 may act as a cofactor of HHV-6 reactivation.

Generation of Cytomegalovirus-Specific Human T-Lymphocyte Clones by Using Autologous B-Lymphoblastoid Cells with Stable Expression of pp65 or IE1 Proteins: a Tool To Study the Fine Specificity of the Antiviral Response

ABSTRACT Cytotoxic T lymphocytes (CTLs) play a central role in the control of persistent human cytomegalovirus (HCMV) infection in healthy virus carriers. Previous analyses of the specificity of HCMV-reactive CD8+ CTLs drawn from in vitro models in which antigen-presenting cells were autologous fibroblasts infected with laboratory HCMV strains have shown focusing of CTL responses against the major tegument protein, pp65. By contrast, the 72-kDa major immediate-early protein (IE1) was identified as a minor target for this response. Here we have studied the fine specificity and T-cell-receptor features of T-cell clones generated against autologous B lymphoblastoid cell lines stably transfected with HCMV cDNA coding for either pp65 or a natural variant of IE1. This strategy allowed efficient generation of T-cell clones against IE1 and pp65 and led to the identification of several new IE1 and pp65 epitopes, including some located in polymorphic regions of IE1. Such an approach may provide relevant information about the characteristics of the CTL response to IE1 and the effect of viral polymorphism on the immune response against HCMV.

A FUT2 Gene Common Polymorphism Determines Resistance to Rotavirus A of the P[8] Genotype

Attachment to carbohydrates of the histo-blood group type of several human Rotavirus strains (RVA) has recently been described. Synthesis of these ligands requires a functional FUT2 enzyme, suggesting that FUT2 null homozygote (ie, nonsecretor) individuals may not be recognized by most human RVA strains. Whereas such individuals represent 20% of the control population, this retrospective study determined that none of 51 patients infected by P[8] rotavirus strains were nonsecretors. The lack of α1,2fucosylated carbohydrate motifs in the gut surface mucosa is thus associated with resistance to symptomatic infection and virus attachment to such motifs is essential to the infection process.

Evidence of recombination in natural populations of hepatitis A virus

Genetic analysis of selected genome regions of hepatitis A virus (HAV) suggested that distinct genotypes of HAV could be found in different geographical regions. At least seven HAV genotypes have been identified all over the world, including four human genotypes (I, II, III, and VII) and three simian strains (IV, V, and VI). Phylogenetic analysis using full-length VP1 sequences revealed that human strain 9F94 has a close genetic relation with strain SLF-88 (sub-genotype VII). Nevertheless, the same analysis using full-length VP2 or VP3 sequences revealed that strain 9F94 has a close genetic relation with strain MBB (sub-genotype IB). To test the possibility of genetic recombination, phylogenetic studies were carried out, revealing that a crossing over had taken place in the VP1 capsid protein. These findings indicate that capsid-recombination can play a significant role in shaping the genetic diversity of HAV and, as such, can have important implications for its evolution, biology, and control.

Features of Epstein-Barr Virus (EBV) reactivation after reduced intensity conditioning allogeneic hematopoietic stem cell transplantation

This single centre study assessed the incidence, kinetics and predictive factors of Epstein-Barr Virus (EBV) reactivation and EBV-related lymphoproliferative diseases (LPDs) in 175 consecutive patients who received a reduced-intensity conditioning (RIC) before allogeneic hematopoietic stem cell transplantation (allo-HSCT). The cumulative incidence of EBV reactivation at 6 months after allo-HSCT defined as an EBV PCR load above 1000 copies of EBV DNA/105 cells was 15%, and none of these patients experienced any sign or symptom of LPD. A total of 17 patients, who had EBV DNA levels exceeding 1000 copies/105 cells on two or more occasions, were pre-emptively treated with rituximab. With a median follow-up of 655 (range, 92–1542) days post allo-HSCT, there was no statistically significant difference in term of outcome between those patients who experienced an EBV reactivation and those who did not. In multivariate analysis, the use of antithymocyte globulin as part of the RIC regimen was the only independent risk factor associated with EBV reactivation (relative risk=4.9; 95% confidence interval, 1.1–21.0; P=0.03). We conclude that patients undergoing RIC allo-HSCT using anti-thymocyte globulin as part of the preparative regimen are at higher risk for EBV reactivation. However, this did not impact on outcome, as quantitative monitoring of EBV viral load by PCR and preemptive rituximab therapy allowed for significantly reducing the risk of EBV-related LPD.

Resistance pattern of cytomegalovirus (CMV) after oral valganciclovir therapy in transplant recipients at high-risk for CMV infection.

In transplant recipients, cytomegalovirus (CMV) resistance to antivirals causes an increasing problem. Here we report the clinical, therapeutic, and virological characteristics of 11 cases of CMV resistance among transplant recipients at high-risk for CMV infection and receiving valganciclovir as a prophylactic, preemptive or maintenance therapy. Active CMV infection was monitored by viral DNA quantification in whole blood, and CMV resistance was assessed by UL97 and UL54 viral gene sequencing. For 10 patients, ganciclovir resistance detected after valganciclovir therapy was associated with one mutation within UL97 phosphotransferase located at codons 460 and 592-603, which constitutes a similar pattern of resistance to what has been reported previously in AIDS patients treated with valganciclovir. For the last patient, two mutations in UL97 and UL54 genes were identified. The start of valganciclovir maintenance treatment after an intravenous curative treatment while CMV DNA is still detectable in peripheral blood might represent a risk factor for the emergence of CMV resistance. The possible emergence of CMV resistance in transplant recipients at high-risk for CMV infection who receive valganciclovir therapy should be taken into account. Among those patients, CMV infection has to be closely monitored in order to detect promptly the emergence of drug-resistance.

Amplified NKG2C+ NK Cells in Cytomegalovirus (CMV) Infection Preferentially Express Killer Cell Ig-like Receptor 2DL: Functional Impact in Controlling CMV-Infected Dendritic Cells

CMV infection represents a major complication in hematopoietic stem cell transplantation, which compromises graft outcome. Downregulation of HLA class I expression is one mechanism by which CMV evades T cell–mediated immune detection, rendering infected cells vulnerable to killer cell Ig-like receptor (KIR)+ NK cells. In this study, we observed that the amplified NKG2C+ NK cell population observed specifically in CMV seropositive individuals mainly expressed KIR2DL receptors. We have shown that HLA class I expression was downregulated on CMV-infected immature dendritic cells (iDCs), which escape to HLA-A2-pp65–specific T lymphocytes but strongly trigger the degranulation of KIR2D+ NK cells. CMV infection conferred a vulnerability of C2C2+ iDCs to educated KIR2DL1+ and KIR2DL3+ NK cell subsets. Alloreactivity of KIR2DL1+ NK cell subsets against C1C1+ iDCs was maintained independently of CMV infection. Unexpectedly, CMV-infected C1C1+ iDCs did not activate KIR2DL3+ NK cell reactivity, suggesting a potential CMV evasion to KIR2DL3 NK cell recognition. Altogether, the coexpression of KIR and NKG2C on expanded NK cell subsets could be related to a functional contribution of KIR in CMV infection and should be investigated in hematopoietic stem cell transplantation, in which the beneficial impact of CMV infection has been reported on the graft-versus-leukemia effect.

Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre-emptive therapy.

Two quantitative duplex real‐time PCR assays were developed for co‐amplification of human albumin and cytomegalovirus (CMV) or Epstein Barr virus (EBV) genes after automated extraction on whole blood, and compared two units for expressing viral DNA loads (copies per ml of blood or per 106 peripheral blood leukocytes (PBLs)) on 1,138 positive samples. Both PCRs were characterized by high sensitivity, reproducibility, and linear range. Automated extraction by a MagNA Pure LC Instrument was shown to be more efficient when peripheral blood cell count was inferior to 5 × 109 PBLs/L. Albumin co‐amplification allows the detection of PCR inhibitors and normalization of viral load according to the number of cells calculated in the sample. The two ways of expressing viral load results were highly correlated, but quantitative differences varied in relation to variations of blood cell count. As these two viruses are highly cell associated, viral loads can be underestimated in patients with leucopenia. In the setting of pre‐emptive strategies during CMV infection, the units in which results are expressed can influence clinical management, as illustrated in this article. J. Med. Virol. 81:90–98, 2009.

Detection of herpes simplex virus (1 and 2), varicella-zoster virus, cytomegalovirus, human herpesvirus 6 and enterovirus in immunocompetent Tunisian patients with acute neuromeningeal disorder.

Enteroviruses (EVs) and human herpesviruses (HHVs) are involved frequently in acute neurological disorders of viral etiology. This study aimed to investigate the incidence of herpes simplex virus types‐1 (HSV‐1) and 2 (HSV‐2), varicella‐zoster virus (VZV), cytomegalovirus (CMV), human herpesvirus 6 (HHV‐6) and human enteroviruses (EVs) in cerebrospinal fluid (CSF) samples of Tunisian immunocompetent patients with neuromeningeal disorders. The patients had been hospitalized at the Fattouma Bourguiba University Hospital (Monastir, Tunisia) between September 2007 and June 2009. At least one viral genome was detected in 58 (46%) out of 126 CSF samples collected. Enterovirus was detected in 31 of the positive samples (53.4%), CMV in 20 (34.5%), HSV‐1 in 3 (5.2%), HSV‐2 in 6 (10.3%), VZV in 4 (6.9%), HHV‐6 in 2 (3.4%). More than one viral genome was detected in seven CSF samples, including CMV DNA in six of the samples. The high frequency of enteroviral infections in aseptic meningitis was confirmed. The detection of CMV DNA only suggests a direct role of this virus in the etiology of acute neuromeningeal disorder. J. Med. Virol. 84:282–289, 2012.

Modulation of HLA-A*0201-Restricted T Cell Responses by Natural Polymorphism in the IE1315–324 Epitope of Human Cytomegalovirus

Cytotoxic T lymphocytes play a central role in the control of persistent human CMV (HCMV) infection and reactivation. In healthy virus carriers, the specific CD8+ CTL response is almost entirely directed against the virion tegument protein pp65 and/or the 72-kDa major immediate early protein, IE1. Studies that included a large panel of HCMV+ donors suggested that immunorelevance of pp65 and IE1 was directly related with individual HLA haplotype difference. Nevertheless, there are no data on the incidence of HCMV natural polymorphism on virus-specific CTL responses. To assess the impact of IE1 polymorphism on CTL response, we have sequenced in 103 clinical isolates the DNA region corresponding to IE1315–324, an immunodominant epitope presented by HLA-A*0201 molecules. Seven peptidic variants were found with extensive difference in their frequencies. The response of four HLA-A*0201-restricted anti-IE1 T lymphocyte clones, which were previously generated from one donor against autologous B lymphoblastoid cells expressing a recombinant clinical variant of IE1, was then evaluated using target cells loaded with mutant synthetic peptides or expressing rIE1 variants. One of four clones, which have been sorted 19 times among 22 clones targeted against IE1315–324, recognized six of the seven tested variant epitopes. All three other clones showed distinct reactivity patterns to target cells loaded with the different mutant peptides or expressing IE1 variants. Therefore, in the HLA-A2 context, clonal expansions of anti-IE1 memory CTLs may confer a protection against HCMV successive infections and reactivations by killing cells presenting most of the naturally occurring IE1315–324 epitope variants.