Variabilité génétique des souches virales HBV et HDV circulant dans la région du Sahara en Afrique et étude de la co-spéciation HBV/HDV

L infection par le virus de l hépatite B (HBV) constitue en Afrique Subsaharienne un problème de santé publique majeur. Le taux de prévalence de la protéine d enveloppe du virus, l antigène HBs (AgHBs) peut atteindre jusqu à 30% dans certains pays. De plus on estime entre 70 à 100 millions le nombre de porteurs chroniques de l HBV avec une fréquence de décès annuels de l ordre de 250 000. Les données concernant l infection concomitante par le virus de l hépatite D (HDV) virus satellite de l HBV, sont très rares car très peu d études ont été conduites. Les génotypes HBV/E, et l HBV/A ont été identifiés en Afrique subsaharienne, le génotype D étant cantonné à l Afrique du Nord. De plus, plusieurs souches recombinantes entre le génotype E et les génotypes, A et D ont aussi été décrit. Concernant l HDV, 4 génotypes africains , HDV 5, -6, -7 et -8 ont été caractérisés au laboratoire chez des patients africains immigrés en France, infectés dans leur pays d origine. Au cours de cette étude nous avons voulu déterminer l épidémiologie moléculaire des souches HBV et HDV circulant au Niger, et plus généralement dans la région du Sahara (Mali de Mauritanie et du Tchad). Au partir d une cohorte de donneurs de sang du Niger porteurs de l AgHBs, nous avons retrouvé que 80% des souches étudiées appartenaient au génotype E. Ces souches présentaient une variabilité génétique significativement plus différente que celle décrite pour les souches HBV/E de la littérature (p<0,005) suggérant une diffusion plus ancienne de l infection au Niger. De plus, nous avons mis en évidence un nouveau recombinant HBV/D-E entre des souches HBV/D et HBV/E, représentant près de 20% des souches isolées de notre cohorte, présentant des points de cassures précis, situés dans des points chauds de recombinaison décrits dans la littérature. Ce recombinant HBV/D-E présentait un taux de divergence dans sa séquence nucléotidique complète de plus de 4% en par rapport aux sous génotypes HBV/D décrits à ce jour. Les analyses phylogénétiques extensives effectuées nous permettent de le classer clairement comme un nouveau sous génotype, nous avons proposé HBV/D8. De même, comme décrits aussi par d autres équipes, nous avons mis en évidence d autres recombinants HBV-E/D, à la fois au Niger, mais aussi en Mauritanie avec des profils différents les uns des autres, témoignant de la grande variabilité génétique des souches virales dans la région. En revanche, la prévalence de l infection Delta au Niger semblait a priori faible. Quatre souches de notre cohorte (7,8%), toutes de génotype HDV-1 ont été isolées. L étude de la co-spéciation HBV/HDV dans cette région de l Afrique saharienne (Niger, Mali de Mauritanie et du Tchad) a été entreprise à partir de 82 échantillons de la collection des sérums HDV positifs du laboratoire. Le génotype E était associé à tous les génotypes delta présents HDV-1, -5 et -7. De même, une souche HBV/D était aussi capable de s associer à l HDV-1 et -5. Afin de tester si l enveloppement de HDV par HBV était dépendant ou non des génotypes des souches virales, nous avons mis au point un modèle cellulaire in vitro de co-transfection transitoire de plasmides codant la protéine AgHBs et la grande protéine delta. La méthode de mesure consistait en l évaluation de la formation de particules pseudo-virales. Les résultats préliminaires obtenus à l aide de HBV/D co-transfecté avec les génotypes HDV-1, HDV-3, HDV-5 et HDV-6 et HDV-7, montrent que HDV-1, mais pas HDV-5, était enveloppé. Grâce à ce modèle, les études seront poursuivies afin d analyser la capacité d enveloppement des différents génotypes delta africains par le génotype E.Infection with hepatitis B (HBV) in SubSaharan Africe is an issue of major public health. The prevalence of the envelope protein of the virus, HBs antigen (HBsAg) can reach up to 30% in some countries. In addition it is estimated between 70 to 100 million, the number of chronic carriers of HBV with an annual death rate of about 250 000. Data on coinfection with hepatitis D (HDV) virus satellite of HBV are very rare because very few studies have been conducted. In terms of molecular characterization of HBV and HDV circulating strains, studies, although partial and conducted with a small number of samples, have been reported. Two HBV genotypes, HBV/E, and HBV/A (with its sub genotypes A1, A2, A3, A4 and A5) have been mainly identified in sub-Saharan Africa. Genotype D is confined to North Africa . In addition, several recombinant strains between genotype E and genotypes A and/or D have also been described. Concerning the HDV, 4 "African genotypes", HDV-5, -6, -7 and -8 have been characterized in the laboratory from African patients immigrants in France, who had been infected in their country of origin. Two studies conducted in Gabon confirmed the presence of HDV genotype-7 and -8. In this study we wanted to determine the molecular epidemiology of HBV and HDV strains circulating in Niger and more generally in the Sahara region, in neighboring countries of Mali from Mauritania and Chad. In a cohort from blood donors in Niger HBsAg carriers, we found that 80% of the studied strains belonged to genotype E. These strains showed genetic variability significantly different from that described for HBV/E strains of the literature (p <0.005) suggesting an ancient diffusion of infection in Niger. Furthermore, we identified a new recombinant HB /D-E between strains HBV/D and HBV / E, representing nearly 20% of strains isolated in our cohort, with the specific breakpoints located in hotspots recombination described elsewhere in the literature. The recombinant HBV/D-E showed a divergence in its complete nucleotide sequence of more than 4% as compared to HBV genotypes /D described to date. The extensive phylogenetic analyses carried out allow us to classify it as clear as a new genotype, we proposed HBV/D8. Similarly, as also described by other teams, we have highlighted other recombinant HBV/E-D, both in Niger, but also in Mauritania with profiles different from each other, reflecting the high genetic variability of viral strains in the region. In contrast, the prevalence of HDV infection in Niger seemed a priori low. Four strains in our cohort (7.8%), all classified as genotype HDV-1 were isolated. The study of HBV / HDV co-speciation in this region of Saharan Africa (Niger, Mali, Mauritania and Chad) was undertaken from 82 samples from the laboratory collection of HDV positive serum. Genotype E was associated with all delta genotypes found, HDV -1, -5 and -7. Similarly, HBV/D strain was also able to envelope the HDV-1 and -5. To test whether the envelopment of HDV or HBV was dependent or not on genotypes of virus strains, we developed a cellular in vitro model of transient co-transfection of plasmids encoding the HBsAg protein and large delta protein. The measurement method consisted of evaluating the formation of viral like particles. Preliminary results obtained with HBV/D co-transfected with HDV-1, -3, -5, -6, and -7, showed that HDV-1, but not HDV-5, was wrapped. With this model, studies are continuing to analyze the ability of the genotype E to wrapping different "Delta African genotypes".PARIS13-BU Sciences (930792102) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

AIDS Res Hum Retroviruses

Immunorecovery could be attenuated in HIV-hepatitis B virus (HBV) co-infection versus HIV mono-infection during antiretroviral therapy (ART), yet whether it also occurs in individuals from Sub-Saharan Africa without severe co-morbidities is unknown. In this study, 808 HIV-infected patients in Cote d'Ivoire initiating continuous ART were included. Six-month CD4+ count trajectories and the proportion reaching CD4+ T-cell counts >350/mm3, HIV-RNA 104 copies/mL). Co-infected patients with high HBV-DNA replication initiated ART with significantly lower median CD4+ T-cell counts (216/mm3, IQR=150-286) compared to co-infection with low HBV-DNA replication (268/mm3, IQR=178-375) or HIV mono-infection (257/mm3, IQR=194-329) (p=0.003). These patients had significantly faster rates of CD4+ cell count increase from baseline after adjusting for baseline age, WHO stage III/IV and CD4+ cell counts (p=0.04), yet were not more likely to exhibit optimal immunorecovery (82.5% versus 80.0% and 77.9%, respectively) (p=0.8). In conclusion, change in CD4+ cell counts after ART-initiation was accelerated in co-infected patients with high HBV DNA viral loads, but this did not lead to increased rates of optimal immunorecovery

Hepatitis B and hepatitis D virus infections in the Central African Republic, twenty-five years after a fulminant hepatitis outbreak, indicate continuing spread in asymptomatic young adults

<div><p>Hepatitis delta virus (HDV) increases morbidity in Hepatitis B virus (HBV)-infected patients. In the mid-eighties, an outbreak of HDV fulminant hepatitis (FH) in the Central African Republic (CAR) killed 88% of patients hospitalized in Bangui. We evaluated infections with HBV and HDV among students and pregnant women, 25 years after the fulminant hepatitis (FH) outbreak to determine (i) the prevalence of HBV and HDV infection in this population, (ii) the clinical risk factors for HBV and/or HDV infections, and (iii) to characterize and compare the strains from the FH outbreak in the 1980s to the 2010 HBV–HDV strains. We performed a cross sectional study with historical comparison on FH-stored samples (n = 179) from 159 patients and dried blood-spots from volunteer students and pregnant women groups (n = 2172). We analyzed risk factors potentially associated with HBV and HDV. Previous HBV infection (presence of anti-HBc) occurred in 345/1290 students (26.7%) and 186/870 pregnant women (21.4%)(<i>p = 0</i>.<i>005</i>), including 110 students (8.8%) and 71 pregnant women (8.2%), who were also HBsAg-positive (<i>p = 0</i>.<i>824</i>). HDV infection occurred more frequently in pregnant women (n = 13; 18.8%) than students (n = 6; 5.4%) (<i>p = 0</i>.<i>010</i>). Infection in childhood was probably the main HBV risk factor. The risk factors for HDV infection were age (<i>p = 0</i>.<i>040</i>), transfusion (<i>p = 0</i>.<i>039</i>), and a tendency for tattooing (<i>p = 0</i>.<i>055</i>) and absence of condom use (<i>p = 0</i>.<i>049</i>). HBV-E and HDV-1 were highly prevalent during both the FH outbreak and the 2010 screening project. For historical samples, due to storage conditions and despite several attempts, we could only obtain partial HDV amplification representing 25% of the full-length genome. The HDV-1 mid-eighties FH-strains did not form a specific clade and were affiliated to two different HDV-1 African subgenotypes, one of which also includes the 2010 HDV-1 strains. In the Central African Republic, these findings indicate a high prevalence of previous and current HBV-E and HDV-1 infections both in the mid-eighties fulminant hepatitis outbreak and among asymptomatic young adults in 2010, and reinforce the need for universal HBV vaccination and the prevention of HDV transmission among HBsAg-positive patients through blood or sexual routes.</p></div

Clinical Outcomes during Treatment Interruptions in Human Immunodeficiency Virus-Hepatitis B Virus Co-infected Patients from Sub-Saharan Africa

Antiretroviral treatment (ART) interruptions increase the risk of severe morbidity/mortality in human immunodeficiency virus (HIV)-infected individuals from subSaharan Africa. We aimed to determine whether the risk is further increased among HIV-hepatitis B virus (HBV) co-infected patients in this setting. In this sub-analysis of a randomized-control trial, 632 participants from Côte d'Ivoire randomized to receive continuous-ART (C-ART), structured ART interruptions of 2-months off, 4-months on (2/4-ART), and CD4-guided ART interruptions (CD4GT, interruption at 350/mm3 and reintroduction at 250/mm3) were analyzed. Incidence rates (IR) of serious HIV- and non-HIV-related morbidity were compared between patients stratified on hepatitis B surface antigen (HBsAg) status. Overall, 65 (10.3%) were HBsAg-positive, 29 (44.6%) of whom had HBV-DNA levels > 10,000 copies/mL. After a median 2.0 year (range = 0.2-3.1) followup, 3 1 serious HIV-related events occurred in 101 HIV mono-infected and 15 HIV-HBV co-infected patients (IR = 10.0 versus 13.2/100 person/years, respectively, P = 0.3), whereas the highest incidence was observed in co-infected patients with baseline HBV-replication > 10,000 copies/mL (IR = 24.0/100 person/years, P versus HIV mono-infected = 0.002). Incidence of bacterial infections was also highest in the co-infected group with HBV-replication > 10,000 copies/mL (IR = 12.9 versus 3.3/100 person/years in HIV mono-infected patients, P = 0.001). The relative effect of CD4GT or 2/4-ART versus C-ART was not different between infection groups (P for interaction = 0.4). No increase in the incidence of non-HIV-related morbidity was observed for co-infected patients (P = 0.5), even at HBV-replication levels > 10,000 copies/mL (P = 0.7). In conclusion, co-infected patients with elevated HBV-replication at ART-initiation are more susceptible to HIV-related morbidity, especially invasive bacterial diseases, during treatment interruption

Comparison of HBV status among young asymptomatic adults (students and pregnant women) in Bangui, Central Africa Republic (CAR) in 2010.

<p>Comparison of HBV status among young asymptomatic adults (students and pregnant women) in Bangui, Central Africa Republic (CAR) in 2010.</p

Alignment of the L-HDAg amino acid deduced sequences from fulminant-associated isolates from the historical cohort (1985) and from young asymptomatic HDV-infected students (2010).

<p>The alignment is compared together with L-HDAg sequences from HDV-1 prototype (Italy, Accession Number:X04451) and HDV-3 prototype (Peru-1, Accession Number:L22063), representing the prototypes of two HDV genotypes associated with fulminant hepatitis outbreaks. Dots represent the same amino acid as in the Italy prototype and question mark ambiguities. A: Full-length coding sequences (214 codons) were obtained for 3 FH isolates (FH27, FH88, FH123). B: COOH terminal part of L-HDAg of the corresponding HDV 1985 (45 FH clones) associated with fulminant hepatitis and 2010 (seven) sequences associated to asymptomatic infections. Note that all the African sequences have the A202S mutation and that FH27 clone 4 has a frameshift mutation of the carboxy-terminal end of L-HDAg (ORF-K), leading to the disappearance of the farnesylation CXXX box.</p

Comparison of HDV status among 182 HBsAg-positive young asymptomatic adults (students and pregnant women) in Bangui, Central Africa Republic (CAR) in 2010.

<p>Comparison of HDV status among 182 HBsAg-positive young asymptomatic adults (students and pregnant women) in Bangui, Central Africa Republic (CAR) in 2010.</p

Phylogenetic analysis of HDV partial genome (<i>400 bp</i>) comparing mid-eighties delta fulminant hepatitis clones (FH1985) to HDV direct sequences (CAR2010) sampled in 2010 from serum (s) and dry blood spot (d) among asymptomatic young adults in Bangui.

<p>We aligned 45 cloned sequences from the 12 FH strains obtained from the historical cohort (labelled ‘1985’ in green) and the 6 strains obtained from asymptomatic students in 2010 represented in duplicate from serum (s) and dried blood (d) (labelled ‘2010’ in blue). We also included 1 strain from a hospitalized case of acute HDV hepatitis in Bangui in 2010 (sd525-CAR2010) and HDV-1 sequences from 9 African samples characterized in Bobigny (dFr) or Lyon (dLy), France, in addition to CAR HDV sequences published by Andernach and coworkers [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006377#pntd.0006377.ref022" target="_blank">22</a>], sampled in 2009. Further comparison included HDV strains from various parts of the world and genotype-reference prototypes (labelled in red). Bayesian analyses (10M generations) gave the consensus tree represented in Fig 1, after discarding 25% of trees from early topology exploration. Branch values indicate posterior probabilities >0.9. Interestingly, the fulminant 1985 and asymptomatic student 2010 strains are all affiliated to HDV-1 with a 100% posterior probability value (thick branch) and the clade topology do not distinguish the mid-eighty strains from the 2010 strains.</p

Prevalence of HBV and HDV markers in 2172 young asymptomatic adults (students and pregnant women) in Bangui (CAR), in year 2010.

<p>Historical cohort (n = 159) corresponds to -20°C cryopreserved serum from a hepatitis delta fulminant outbreak and control samples collected in the mid-eighties. Data are mean (SD), or n (%) unless otherwise stated. ND, not determined.</p