Céline Bressollette-Bodin

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.

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.

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.

Both the nature of KIR3DL1 alleles and the KIR3DL1/S1 allele combination affect the KIR3DL1 NK-cell repertoire in the French population

NK‐cell functions are regulated by many activating and inhibitory receptors including KIR3DL1. Extensive allelic polymorphism and variability in expression can directly alter NK‐cell phenotype and functions. Here we investigated the KIR3DL1+ NK‐cell repertoire, taking into account the allelic KIR3DL1/S1 polymorphism, KIR3DL1 phenotype, and function. All 109 studied individuals possessed at least one KIR3DL1 allele, with weak KIR3DL1*054, or null alleles being frequently present. In KIR3DL1high/null individuals, we observed a bimodal distribution of KIR3DL1+ NK cells identified by a different KIR3DL1 expression level and cell frequency regardless of a similar amount of both KIR3DL1 transcripts, HLA background, or KIR2D expression. However, this bimodal distribution can be explained by a functional selection following a hierarchy of KIR3DL1 receptors. The higher expression of KIR3DL1 observed on cord blood NK cells suggests the expression of the functional KIR3DL1*004 receptors. Thus, the low amplification of KIR3DL1high, KIR3DL1*004 NK‐cell subsets during development may be due to extensive signaling via these two receptors. Albeit in a nonexclusive manner, individual immunological experience may contribute to shaping the KIR3DL1 NK‐cell repertoire. Together, this study provides new insight into the mechanisms regulating the KIR3DL1 NK‐cell repertoire.

Herpesvirus detection and cytokine levels (IL-10, IL-6, and IFN-γ) in ocular fluid from Tunisian immunocompetent patients with uveitis

Human herpesviruses (HHVs) are involved in the pathogenesis of different types of uveitis. Cytokine response plays an important role in virus‐induced immunopathology. This study aimed to investigate the incidence of HHVs in aqueous humor samples of immunocompetent patients with suspected viral uveitis and cytokine (IL‐10, IL‐6, and IFN‐γ) expression profiling. Forty‐seven aqueous humor samples were collected from immunocompetent patients with viral uveitis. Samples were assayed for HHV‐1 to HHV‐8 by in‐house real‐time polymerase chain reactions. IL‐6, IL‐10, and IFN‐γ were quantified with a cytometric bead array. Relations between viral detection, cytokine profiles, and clinical data were studied. At least one viral genome was detected in 21 aqueous humor samples analyzed. Varicella‐zoster virus (VZV) was detected in 14 of the positive samples, cytomegalovirus (CMV) in 8, HSV‐1 in 1, Epstein–Barr virus (EBV) in 4, and HHV‐6 in 2. More than one viral genome was detected in seven aqueous humor samples. Aqueous humor samples positive for HHV‐DNA contained significant levels of IL‐6, IL‐10, and IFN‐γ, compared to HHV‐DNA negative samples. High levels of IL‐6 were detected in patients with CMV‐DNA in their aqueous humor samples. Significantly higher levels of IL‐10 and IFN‐γ were found in positive samples for VZV, EBV, and HHV‐6 DNA than in negative aqueous humor ones. VZV was the principal etiologic agent of uveitis in this Tunisian series, with CMV the second most common agent. Knowledge of immunoregulatory interactions and dynamic changes in viral uveitis may be a key to understand the pathogenesis leading to more‐effective treatments. J. Med. Virol. 85:2079–2086, 2013.

Long-lasting HHV-6 reactivation in long-term adult survivors after double umbilical cord blood allogeneic stem cell transplantation

Higher incidence of human herpesvirus 6 (HHV-6) infection has been documented after umbilical cord blood allo-transplant in adults. Here we demonstrate that HHV-6 reactivation persists for a very long time in half of the patients after this type of graft. Long-term immune reconstitution does not explain this event, which remains to be explained.

MICA Mutant A5.1 Influences BK Polyomavirus Reactivation and Associated Nephropathy After Kidney Transplantation

BACKGROUND BK polyomavirus (BKPyV) frequently reactivates in kidney transplant recipients during immunosuppressive therapy and triggers BKPyV-associated nephropathy and graft rejection. Determining effective risk factors for BKPyV reactivation is required to achieve efficient prevention. METHODS This study investigated the role of major histocompatibility complex (MHC) class I-related chain A (MICA) in BKPyV reactivation in a cohort of 144 transplant donor/recipient pairs, including recipients with no reactivation (controllers) and those with mild (virurics) or severe (viremics) BKPyV reactivation after graft receipt. RESULTS We show that, in the kidney, MICA is predominantly expressed in tubule epithelial cells, the natural targets of BKPyV, questioning a role for MICA in the immune control of BKPyV infection. Focusing on MICA genotype, we found a lower incidence of BKPyV reactivation in recipients of a renal graft from a donor carrying the MICA A5.1 mutant, which encodes a truncated nonconventional MICA. We established that a mismatch for MICA A5.1 between transplant donor and recipient is critical for BKPyV reactivation and BKPyV-associated nephropathy. Functionally, we found that a low prevalence of BKPyV reactivation was associated with elevated anti-MICA sensitization and reduced plasma level of soluble MICA in recipients, 2 potential effector mechanisms. DISCUSSIONS These findings identify the MHC-related MICA as an immunogenetic factor that may functionally influence anti-BKPyV immune responses and infection outcomes.

Impact of stem cell graft on early viral infections and immune reconstitution after allogeneic transplantation in adults

BACKGROUND Viral infections are well-known complications after allogeneic stem cell transplant (allo-SCT). OBJECTIVES We compared prospectively incidences of DNAemia and active infections (AI) for five opportunistic viruses (Human Herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), BK polyomavirus (BKPyV), Cytomegalovirus (CMV) and Adenovirus (ADV)) and kinetics of immune reconstitution (IR) in adults receiving either double umbilical cord blood (dUCB group) or unrelated peripheral blood stem cell (uPBSC group) allo-SCT after a reduced-intensity conditioning (RIC) regimen. STUDY DESIGN Whole blood samples were collected at transplant, every 15days during the first 3 months and at 4, 5 and 6 months post-transplant. RESULTS Sixty-five patients were enrolled (uPBSC n=34; dUCB n=31). Incidences of HHV-6 and BKPyV DNAemia were significantly higher for dUCB (97% vs 23.5% and 58% vs 32%, respectively) while EBV DNAemia was more frequently detected in uPBSC (71% vs 26%). The incidence of CMV DNAemia was similar between both groups. ADV AI developed only in dUCB. HHV-6 AI were also higher in dUCB (84% vs 21%). In multivariate analysis, dUCB graft was the only independent factor associated with HHV-6 DNAemia (OR: 19.0; 95%CI: 5.2-69.1; p<0.0001) while EBV DNAemia were significantly associated with uPBSC (OR: 29.9; 95%CI: 5.68-158; p <0.0001). dUCB graft was also the only factor associated with HHV-6 AI. Finally, higher counts and faster recoveries of B lymphocytes (p<0.0001) and monocytes (p=0.0007) were observed in the dUCB group. CONCLUSION We demonstrate a strong correlation between sources of graft and patterns of viral DNAemia and AI and IR after RIC allo-SCT.

Quantification of two viral transcripts by real time PCR to investigate human herpesvirus type 6 active infection

BACKGROUND Human herpesvirus 6 (HHV-6) causes exanthema subitum and is associated with symptomatic reactivations in immunocompromised patients, particularly after hematopoietic stem cell transplantation. The detection of viral mRNA can help to make the difference between latent, chromosomally integrated and true replicating virus. It can also be a useful tool to investigate viral multiplication in different cell types. OBJECTIVES To develop molecular tools for the detection and quantification HHV-6 transcripts that can be used in a clinical setting. STUDY-DESIGN Two one-step reverse-transcriptase quantitative real-time PCR (RT-qPCR) were developed for the quantification of the immediate early U90 and the late U100 mRNAs. Viral mRNA loads were compared to viral DNA loads during infection in vitro and in blood samples collected from stem cell transplanted patients. RESULTS Analytical performances of the two quantitative real-time PCR were good. In vitro, kinetics of both transcripts was well correlated with DNA levels. Sixty blood samples from patients with active HHV-6 infection were analyzed. Overall agreement of qualitative results for HHV-6 DNA, U90 RNA and U100 RNA was good. HHV-6 DNA loads were significantly higher than mRNA loads. In clinical samples, the amounts of U100 and U90 mRNAs were low and their detection was mainly associated to viral DNA loads upper than 1000 copies/ml of blood. CONCLUSION The new assays are sensitive and reliable methods for the monitoring of viral transcription in vitro and in vivo. As their detection is associated to high DNA loads in vivo, they can be helpful tools for the diagnosis of active infection.