Occult Hepatitis B Virus Infection and Liver Fibrosis in Chinese Patients.

BACKGROUND: The impact of hepatitis B surface antigen (HBsAg)-negative/hepatitis B virus (HBV) DNA-positive occult HBV infection (OBI) on the severity of liver fibrosis remains unclear. METHODS: A total of 1772 patients negative for HBsAg but positive for antibody to hepatitis B core antigen (HBcAg), stratified by the presence or absence of OBI, were selected for long-term carriage leading to elevation of ≥2 of 4 liver fibrosis indexes-hyaluronic acid (HA), laminin, type III procollagen peptide (PCIII), and type IV collagen (CIV)-at testing in a Chinese hospital. Patients were tested for serum viral load, HBV markers, and histopathological changes in liver biopsy specimens. RESULTS: OBI was identified in 148 patients with liver fibrosis (8.4%), who had significantly higher levels of HA, laminin, PCIII, and CIV than 1624 fibrotic patients without OBI (P < .05). In 36 patients with OBI who underwent liver biopsy, significant correlations were observed between OBI viral load and serum HA levels (P = .01), PCIII levels (P = .01), and pathological histological activity index (HAI) scores (P < .001), respectively; HAI scores and PCIII levels (P = .04); HBcAg immunohistochemical scores and HA levels (P < .001); and HBcAg immunohistochemical scores and PCIII levels (P = .03). Positive fluorescent in situ hybridization results were significantly more frequent in patients with OBIs (80.6% vs 37.5% in those without OBIs). Among patients with OBIs, HBcAg was detected in the liver tissue in 52.8% and HBsAg in 5.6%. CONCLUSIONS: OBI status appears to be associated with liver fibrosis severity.

Effectiveness of the HCV blood screening strategy through eighteen years of surveillance of HCV infection in blood donors in France.

BACKGROUND: The question of maintaining blood screening based on both Hepatitis C virus (HCV) infection antibodies (Ab) and Nucleic Acid Testing (NAT) has been raised in several countries. The French blood donor surveillance database was used to address this issue. MATERIALS AND METHODS: In France, HCV-NAT was implemented in mini pools (MP) in 2001 and in individual testing (ID) in 2010. HCV-positive donations are further investigated including detection of RNA with an alternative polymerase chain reaction assay: Amplicor HCV v2.0 (Roche; LOD95 50 IU/mL) from 2001 to 2006 and CobasTaqMan (CTM) HCV 2.0 assay (Roche; LOD95 9.3 IU/mL) since 2007. RESULTS: From 2001 to 2018, 3,058/48.8 million donations were confirmed HCV positive: 64.4% were Ab+/NAT+, 35.1% Ab+/NAT- and 0.5% Ab-/NAT+. From 2001 to 2018, the NAT yield decreased from 0.65 per million donations to 0, and NAT+ donations dropped from 77% to 46% of the total of HCV donations. 2,491/3,058 were further tested for HCV-RNA: 1,032 (816 NAT+, 216 NAT-) with Amplicor and 1,459 (897 NAT+, 562 NAT-) with CTM. Four (3 MP and 1 ID-NAT, 0.5%) of the 778 NAT negative donations had low viral loads. DISCUSSION: The decline in HCV-NAT yield cases raises the question of the relevance of NAT. Conversely, the increase in Ab+/NAT-donors, suggesting a growing number of resolved infections, argue for Ab discontinuation. In our experience, at least 0.5% of Ab+/NAT-donations had low RNA level when retested. Although the risk of viral transmission by such donations is probably low, the uncertainty associated with their infectivity goes against the removal of Ab in blood screening in our country.

Role of core protein mutations in the development of occult HBV infection.

BACKGROUND & AIMS: Occult HBV infection (OBI) is associated with transfusion-transmitted HBV infection and hepatocellular carcinoma. Studies on OBI genesis have concentrated on mutations in the S region and the regulatory elements. Herein, we aimed to determine the role of mutations in the core region on OBIs. METHODS: An OBI strain (SZA) carrying 9 amino acid (aa) substitutions in the core protein/capsid (Cp) was selected by sequence alignment and Western blot analysis from 26 genotype B OBI samples to extensively explore the impact of Cp mutations on viral antigen production in vitro and in vivo. RESULTS: A large panel of 30 Cp replicons were generated by a replication-competent pHBV1.3 carrying SZA or wild-type (WT) Cp in a 1.3-fold over-length of HBV genome, in which the various Cp mutants were individually introduced by repairing site mutations of SZA-Cp or creating site mutations of WT-Cp by site-directed mutagenesis. The expression of HBcAg, HBeAg, and HBsAg and viral RNA was quantified from individual SZA and WT Cp mutant replicons in transfected Huh7 cells or infected mice, respectively. An analysis of the effect of Cp mutants on intracellular or extracellular viral protein production indicated that the W62R mutation in Cp had a critical impact on the reduction of HBcAg and HBeAg production during HBV replication, whereas P50H and/or S74G mutations played a limited role in influencing viral protein production invivo. CONCLUSIONS: W62R and its combination mutations in HBV Cp might massively affect HBcAg and HBeAg production during viral replication, which, in turn, might contribute to the occurrence of OBI. LAY SUMMARY: Occult hepatitis B virus infections (OBIs) have been found to be associated with amino acid mutations in the S region of the HBV, but the role of mutations in the core protein (Cp) remains unclear. In this study, an OBI strain (SZA) carrying 9 amino acid substitutions in Cp has been examined comprehensively in vitro and in vivo. The W62R mutation in Cp majorly reduces HBcAg and HBeAg production during HBV replication, potentially contributing to the occurrence of OBI.

Follow-up program for blood donors with unconfirmed screening results reveals a high false-positive rate in Dalian, China.

BACKGROUND: Chinese blood donors with unconfirmed serological and/or molecular screening results are deferred permanently. This study investigated the implementation and performance of a follow-up program aiming to improve the notification and management of deferred donors in Dalian, China. STUDY DESIGN AND METHODS: From January 2013 to February 2018, 411,216 donations were tested for HBsAg, anti-HCV, anti-HIV/HIV antigen, and antibodies to Treponema pallidum. HBV, HCV, and HIV nucleic acid testing (NAT) was performed in mini-pools of six or in individual donations (IDs). Reactive donations were evaluated further with alternative serological assays and ID-NAT re-testing. A follow-up procedure was developed to evaluate a subset of deferred donors that were either potential NAT yield cases, serology non-reactive and NAT non-repeated reactive (NRR), or serology NRR irrespective of NAT result. RESULTS: Serological and molecular routine, plus supplemental testing, identified HBV, HCV, HIV, and TP in 503 (0.12%), 392 (0.09%), 156 (0.04%), and 2041 (0.49%) donations, respectively. Overall, 683 of 4156 (16.4%) eligible donors and 205 donors deferred prior 2013 participated in the program. They included 664 serology NRR and 224 NAT yield cases, and 58.8% repeat donors. All markers combined, follow-up documented false reactivity, primary acute infections, and OBI in 61.9% (550/888), 3.3% (29/888), and 12.8% (114/888) of these donors, respectively. Isolated anti-HBc or anti-HBs reactivity was observed in 22% of cases. CONCLUSION: Follow-up testing refined infection status in 78.0% (693/888) of deferred donors with unconfirmed screening results. This high false-positive rate encouraged to reevaluate the current screening strategies and to consider donor reentry.

Occurrence of occult hepatitis B virus infection associated with envelope protein mutations according to anti-HBs carriage in blood donors.

OBJECTIVES: Occult hepatitis B virus infection (OBI) carries a risk of hepatitis B virus (HBV) transmission and hepatocellular carcinoma. As previous studies have had a limited sample size, the characteristics of OBI with genotype B and C (OBIB and OBIC) mutations relating to hepatitis B surface antibody (anti-HBs) elicited by vaccination or a limited host immune response to HBV have not been fully explored. METHODS: In this study, the occurrence of OBIB or OBIC strains associated with envelope protein (pre-S/S) amino acid substitutions obtained from 99 blood donors stratified according to anti-HBs carriage were characterized extensively. RESULTS: According to the presence of anti-HBs within each genotype, the number and frequency of substitution sites specific for anti-HBs(-) OBIB were higher than those specific for anti-HBs(+) OBIB strains (67 vs 31; 117 vs 41), but the reverse pattern was found in OBIC strains (3 vs 24; 3 vs 26). Mutations pre-s1T68I and sQ129R/L were found uniquely in 15-25% of anti-HBs(+) OBIB carriers and mutation pre-s1A54E was found preferentially in anti-HBs(+) OBIC, while 17 substitutions were found preferentially in 11-38% of anti-HBs(-) OBIB strains. In the major hydrophilic region (MHR) region, mutations sS167 in OBIB, sT118 in OBIC, and sA166 in both genotypes were possibly immune-induced escape mutation sites. CONCLUSIONS: Several mutations in pre-S/S of OBI appeared to be associated with carrier anti-HBs pressure, which might be risk factors for potential reactivation of viruses under anti-HBs selection in OBI carriers.

Poor efficacy of nucleic acid testing in identifying occult HBV infection and consequences for safety of blood supply in Italy.

BACKGROUND & AIMS: In Italy, DNA screening of blood donations for hepatitis B virus (HBV) was introduced to prevent the transmission of window period and occult HBV infection. Anti-HBc screening is not recommended in order to avoid shortage of the blood supply. To contain costs, donor samples are generally pooled before testing. We evaluated the safety of this national policy using a prospective repository of donors/recipient pairs. METHODS: We used highly sensitive nucleic acid testing (NAT) assays to test repository and follow-up samples from donors who were initially classified as negative by minipool NAT assays (6-MP), but were later found to carry occult HBV DNA. When available, we also analysed recipients' pre- and post-transfusion samples, collected in the context of a repository financed by the European Commission (the BOTIA project). RESULTS: Between 2008 and 2011 6-MP NAT assays identified 18 carriers of occult HBV infection among 12,695 donors; 28 samples from previous donations were available from 13 of these carriers. Highly sensitive HBV DNA detection methods showed that 6-MP HBV DNA screening failed to identify 14/28 (50%) viraemic donations, that were released for transfusion. HBV marker testing of such blood product recipients revealed two cases of transfusion transmitted HBV infection, documented by donor-recipient sequence identity. CONCLUSIONS: Viraemic blood donations from occult HBV infection carriers remain undetected by current minipool HBV DNA screening, and transfusion transmission of HBV continues to occur in susceptible patients. More effective individual HBV DNA screening and/or tests for antibodies to HBV core antigen should be considered to improve blood safety.

Comparison of seven hepatitis B virus (HBV) nucleic acid testing assays in selected samples with discrepant HBV marker results from United States blood donors.

BACKGROUND: Sensitive triplex nucleic acid tests (NATs) are implemented for blood donation screening worldwide. Assays have variable ability to detect low-level hepatitis B virus (HBV) DNA. At borderline DNA detection levels, where Poisson distribution impacts results, distinguishing true-positive from false-positive results is challenging. Algorithms are needed to confirm such low-level HBV DNA-positive samples. STUDY DESIGN AND METHODS: A total of 135 blood donor samples reactive by one or more HBV markers that provided discrepant results were tested undiluted with four commercial NATs: Ultrio, Ultrio Plus, MPX, and a quantitative assay (SuperQuant). To further explore discrepancies, three additional in-house NATs including real-time polymerase chain reaction (PCR) and nested PCR and sequencing were performed. RESULTS: The numbers reactive of these 135 "difficult" samples by four commercial NATs were as follows: 39 of 107 (36%) with SuperQuant, 40 (30%) with Ultrio, 100 (74%) with Ultrio Plus, and 102 (76%) with MPX. Of the seven NATs, 109 (81%) samples were reactive by at least two assays and thus considered confirmed positive of which 67 (50%) generated a sequence. Ultrio Plus and MPX performed similarly as above (80%-85% detected of 109 and 81%-90% of 67, respectively). Older (median, 49 years), HBV core antibody-reactive donors carried predominantly Genotype A (58%) with high-frequency amino acid substitutions in the major hydrophilic region of the S-protein. Younger (median, 24 years) hepatitis B surface antigen-positive donors carried wild-type strains predominantly Genotype B (32%) and E (24%), the latter in an apparent cluster. CONCLUSIONS: Highly sensitive NATs require new confirmatory algorithms as presented optimally using different genomic regions or sequence generation. The introduction of immigration-related HBV genotypes may impact HBV epidemiology in the United States.

Specific amino acid substitutions in the S protein prevent its excretion in vitro and may contribute to occult hepatitis B virus infection.

Occult hepatitis B virus (HBV) infection (OBI) is defined as low plasma level of HBV DNA with undetectable HBV surface antigen (HBsAg) outside the preseroconversion window period. The mechanisms leading to OBI remain largely unknown. The potential role of specific amino acid substitutions in the S protein from OBI in HBsAg production and excretion was examined in vitro. HBsAg was quantified in culture supernatants and cell extracts of HuH-7 cells transiently transfected with plasmids containing the S gene of eight HBsAg(+) controls and 18 OBI clones. The intracellular (IC)/extracellular (EC) HBsAg production ratio was ∼1.0 for the majority of controls. Three IC/EC HBsAg patterns were observed in OBI strains clones: pattern 1, an IC/EC ratio of 1.0, was found in 5/18 OBI clones, pattern 2, detectable IC but low or undetectable EC HBsAg (IC/EC, 7.0 to 800), was found in 6/18 OBIs, and pattern 3, low or undetectable IC and EC HBsAg, was found in 7/18 clones. Intracellular immunofluorescence staining showed that in pattern 2, HBsAg was concentrated around the nucleus, suggesting retention in the endoplasmic reticulum. The substitution M75T, Y100S, or P178R was present in 4/6 pattern 2 OBI clones. Site-directed-mutagenesis-corrected mutations reversed HBsAg excretion to pattern 1 and, when introduced into a control clone, induced pattern 2 except for Y100S. In a control and several OBIs, variants of a given quasispecies expressed HBsAg according to different patterns. However, the P178R substitution present in all cloned sequences of two OBI strains may contribute significantly to the OBI phenotype.

Head-to-head comparison of two transcription-mediated amplification assay versions for detection of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus Type 1 in blood donors.

BACKGROUND: The second triplex transcription-mediated amplification (TMA) assay version (Ultrio Plus, Novartis Diagnostics) uses an additional reagent enhancing the disruption of hepatitis B virus (HBV) particles and release of DNA for the target capture probe. This study compares the performance of this new assay version with the previous one (Ultrio). STUDY DESIGN AND METHODS: For analytical sensitivity assessment the World Health Organization HBV, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) international standards and various genotype dilution panels were used. Individual donations (IDs) from 9980 first-time donors were screened simultaneously by serology and both TMA assay versions. RESULTS: The 50 and 95% limits of detection (LODs) for HBV using Ultrio Plus were 0.8 (0.6-1.0) and 4.6 (3.2-7.2) IU/mL, respectively, 2.4 (1.4-4.8)-fold more sensitive than Ultrio. The TMA assay versions had comparable LODs for HIV-1 and HCV. The improvement factors on analytical sensitivity panels of HBV Genotypes A to G ranged from 1.3 to 7.3 and 50% LODs (95% confidence interval) reduced from 12.5 (10-15) to 3.8 (3.2-4.4) copies/mL. One Ultrio Plus HBV Genotype D yield sample missed by the Ultrio assay in the donor screening study was detected with ninefold higher sensitivity. The specificities of ID nucleic acid test (ID-NAT) and serologic testing in a similar repeat test algorithm were 100 and 99.41%, respectively. CONCLUSION: More efficient target capture chemistry in the new TMA assay version significantly improved sensitivity and diminished variability in detecting HBV strains of various genotypes. We recommend a triplicate ID-NAT repeat test strategy to eliminate discriminatory tests on false-non-repeat-reactive (anti-HBc-nonreactive) donations.

Human herpesvirus 8 transfusion transmission in Ghana, an endemic region of West Africa.

BACKGROUND: Human herpesvirus 8 (HHV-8) seroprevalence ranges between less than 5% in Europe and North America and 50% to 70% in sub-Saharan Africa. Evidence of HHV-8 transfusion transmission is only indirect. We conducted a serologic (anti-HHV-8) and molecular (HHV-8 DNA) study of samples from paired donor-immunocompetent recipients transfused with whole blood. STUDY DESIGN AND METHODS: Samples from 252 donor-recipient pairs were tested. Immunoglobulin G to HHV-8 was detected with enzyme immunoassays and confirmed with an in-house immunofluorescence assay. The cellular fraction from seroreactive donors and their recipients was tested for HHV-8 DNA. RESULTS: Anti-HHV-8 was positive (reactive in two or more assays) in 28 (11%) patients and 16 (6%) donors. Of 12 seronegative recipients (at risk of transmission) receiving seropositive blood, one very likely transmission was identified (8.3% confidence interval, 0%-23%). The donor blood contained HHV-8 DNA and his and four other donors' sequences clustered separately from recorded genotypes with a 97% bootstrap constituting a distinct genotype. CONCLUSIONS: HHV-8 is transmitted in Ghana but does not carry clinical consequences since most patients are immunocompetent. The clinical risk will increase with the availability of immunosuppressive drugs in sub-Saharan Africa. We propose that a new genotype (HHV-8-G for Ghana) be added to the current nomenclature.

Hepatitis B virus in transfusion medicine: still a problem?

Hepatitis B virus (HBV) has probably evolved with humans for nearly 35,000 years. HBV diversified into 9 genotypes (A-I) presenting specific features directing epidemiology, clinical expression and testing. Genotypes E and C are more infectious and carry higher risk of chronicity and cancer. HBsAg blood screening implemented 40 years ago enormously decreased the risk of transfusion transmission but the remaining risk requires extremely sensitive nucleic acid testing (NAT) to be removed. Limitations of the host immune system, the impact of immunodeficiency and the mechanisms utilised for viral persistence were recently identified. HBV replication produces excess HBsAg and infectious and defective viral particles but screening assays for HBsAg or viral particles alone do not allow fully efficient detection, making necessary screening for both. The host immune system fails to completely control the virus that escapes and persists unrecognized at very low levels or as immuno-selected variants. Variants may not be identified by assays, explaining false negative results. Specific mutations may affect post-transcriptional mechanisms including HBV RNA splicing. Asymptomatic HBV infected blood donors are at risk of long-term complications through mechanisms to be understood for appropriate counselling. Infectivity of occult HBV infection (OBI) by transfusion appears low, anti-HBc (anti-core antigen) only being more infectious than anti-HBs (anti-S protein) positive units.

Nucleic acid testing to detect HBV infection in blood donors.

BACKGROUND: The detection of hepatitis B virus (HBV) in blood donors is achieved by screening for hepatitis B surface antigen (HBsAg) and for antibodies against hepatitis B core antigen (anti-HBc). However, donors who are positive for HBV DNA are currently not identified during the window period before seroconversion. The current use of nucleic acid testing for detection of the human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNA and HBV DNA in a single triplex assay may provide additional safety. METHODS: We performed nucleic acid testing on 3.7 million blood donations and further evaluated those that were HBV DNA-positive but negative for HBsAg and anti-HBc. We determined the serologic, biochemical, and molecular features of samples that were found to contain only HBV DNA and performed similar analyses of follow-up samples and samples from sexual partners of infected donors. Seronegative HIV and HCV-positive donors were also studied. RESULTS: We identified 9 donors who were positive for HBV DNA (1 in 410,540 donations), including 6 samples from donors who had received the HBV vaccine, in whom subclinical infection had developed and resolved. Of the HBV DNA-positive donors, 4 probably acquired HBV infection from a chronically infected sexual partner. Clinically significant liver injury developed in 2 unvaccinated donors. In 5 of the 6 vaccinated donors, a non-A genotype was identified as the dominant strain, whereas subgenotype A2 (represented in the HBV vaccine) was the dominant strain in unvaccinated donors. Of 75 reactive nucleic acid test results identified in seronegative blood donations, 26 (9 HBV, 15 HCV, and 2 HIV) were confirmed as positive. CONCLUSIONS: Triplex nucleic acid testing detected potentially infectious HBV, along with HIV and HCV, during the window period before seroconversion. HBV vaccination appeared to be protective, with a breakthrough subclinical infection occurring with non-A2 HBV subgenotypes and causing clinically inconsequential outcomes. (Funded by the American Red Cross and others.).

Impact of hepatitis B virus surface protein mutations on the diagnosis of occult hepatitis B virus infection.

UNLABELLED: Genotype D occult hepatitis B virus (HBV) infections (OBIs) have a high frequency of amino acid substitutions in the major hydrophilic region of the small surface protein (S protein). This possibly reflects an escape mutation mechanism to evade detection by the host immune system. Mutations may also impact the detection of hepatitis B surface antigen (HBsAg) by commercial assays. To test these hypotheses, 20 recombinant HBV genotype D surface proteins from OBI carriers with or without antibody to hepatitis B surface antigen (anti-HBs) were expressed in yeast. Recombinant surface protein (rS protein) variants were nonreactive with autologous anti-HBs but reacted weakly with vaccine-induced anti-HBs supporting an immune escape mechanism. rS protein variants tested with a wide range of HBs antibodies, and HBsAg commercial assays showed significantly lower antigenic reactivity in anti-HBs carriers than in donors with antibody to hepatitis B core antigen (anti-HBc) only. Eight out of 10 recombinant variants from anti-HBs carriers reacted weakly or were nonreactive with antibodies to HBs as well as with qualitative and quantitative commercial HBsAg assays, whereas eight out of 10 anti-HBc-only plasmas were fully reactive. rS proteins with substitutions of wild-type cysteine at positions 121, 124, and 137 were nonreactive or showed poor reactivity. However, mutation of cysteine 147 did not alter reactivity compared with controls. Restoration of cysteines 124 and 137 by site-directed mutagenesis improved antigenic reactivity. CONCLUSION: Escape mutation is a mechanism associated with OBI, which also leads to decreased reactivity in HBsAg detection assays. Performance of commercial assays would be improved by the incorporation of OBI mutants in reagent development.

Efficacy of hepatitis B virus (HBV) DNA screening and characterization of acute and occult HBV infections among blood donors from Madrid, Spain.

BACKGROUND: Screening of blood units for hepatitis B virus (HBV) DNA identifies donations collected during the window period (WP) of the acute infection and may improve viral safety of the blood supply. It also leads to the detection of occult hepatitis B infection (OBI). STUDY DESIGN AND METHODS: From January 2005 to December 2006, a total of 383,267 blood units were screened for hepatitis B surface antigen (HBsAg) and HBV DNA in two transfusion centers in Madrid, using either individual-donation nucleic acid testing (ID-NAT) or minipool (MP-NAT) of eight donations (MP8). Samples positive for HBV DNA and negative for HBsAg were confirmed by a second molecular test, the viral DNA was quantified, and a genome fragment including the region encoding the major hydrophilic region (MHR) of HBsAg was sequenced. RESULTS: The overall yield of HBV DNA-positive, HBsAg-negative units was 1 in 21,282 (18 cases), higher when using ID-NAT than MP8-NAT (1:9862 vs. 1:51,011; p < 0.01). Four donations (1/95,817) were collected during the infectious pre-HBsAg WP, one during an early recovery stage, and the remaining 13 (1/29,482) were OBIs, six of whom had no detectable antibody to HBsAg. Low-level Genotype D HBV DNA was detected in all OBI cases; the frequencies of this genotype and MHR amino acid substitutions were significantly higher than reported from unselected Spanish HBsAg carriers. Donors with OBI had normal aminotransferase levels and were significantly older than donors carrying HBsAg. CONCLUSIONS: Blood donors in the WP and with OBI are not uncommon in Madrid and are detected at a higher frequency with ID-NAT than MP-NAT.

Deletions and recombinations in the core region of hepatitis B virus genotype E strains from asymptomatic blood donors in Guinea, west Africa.

The prevalence of hepatitis B virus (HBV) surface antigen (HBsAg) chronic carriage in west Africa is the highest in the world, but its molecular epidemiology remains relatively poorly investigated. Plasma samples from random asymptomatic carriers of HBsAg in Conakry, Guinea, were studied and the complete genome sequences of 81 strains were obtained. Three additional samples from Kumasi, Ghana, were also included in the analysis. Phylogenetic analyses confirmed the dominance of genotype E (95.1%), including 8.6% of strains (viral load, 5x10(3)-2.6x10(8) IU ml(-1)) comprising dominant variants with large deletions in the core region and minority wild-type variants. The presence of two different patterns of deletions in two and four donors suggested targeted genome fragility between nt 1979 and 2314. The remaining sequences included one subgenotype A3 (1%) and six A/E recombinant forms (4-7%). A/E strains with identical points of recombination in three donors suggested strongly that these recombinant HBV strains are circulating and transmitted in the population. Recombination points were concentrated in the core gene. The detection of similar A/E recombinant strains in Ghana suggested a geographical extension of recombinant HBV to the region. The quasispecies of one additional Ghanaian strain sequenced in the pre-surface/surface region resolved into dominant clones of either the A or E genotype, but also three different patterns of A/E recombinant variants. The observation that both deletions of genotype E strains and A/E recombination points are mostly located in the core gene at specific positions indicates a region of the genome where genetic rearrangements preferentially take place.

HIV escape from natural killer cytotoxicity: nef inhibits NKp44L expression on CD4+ T cells.

OBJECTIVE: HIV infection induces a progressive depletion of CD4 T cells. We showed that NKp44L, a cellular ligand for an activating natural killer (NK) receptor, is expressed on CD4 T cells during HIV infection and is correlated with both CD4 cell depletion and increase in viral load. NKp44LCD4 T cells are highly sensitive to the NK lysis activity. In contrast, HIV-infected CD4 T cells are resistant to NK killing, suggesting that HIV-1 developed strategies to avoid detection by the host cell immunity. DESIGN: To assess whether viral protein can affect NKp44L expression, using Nef-deficient virus as well as a panel of recombinant vaccinia viruses expressing all HIV-1 viral proteins was tested. The involvement of Nef in the downmodulation of NKp44L was determined using defined mutants of Nef. Functional consequences of Nef on NK-cell recognition were evaluated by either 51Cr-release assays and degranulation assays in presence of anti-NKp44L mAb. RESULTS: We observed that during HIV-1 infection, noninfected CD4 T cells exclusively expressed NKp44L, and demonstrate that Nef mediates NKp44L intracellular retention in HIV-infected cells. This has functional consequences on HIV-infected CD4 T cells recognition by NK cells, causing a decreased susceptibility to NK cytotoxicity. Furthermore, experiments in presence of neutralizing NKp44L mAb revealed that Nef inhibitory effect on NK cytotoxicity mainly depends on the NKp44L pathway. CONCLUSION: This novel escape mechanism could explain the resistance of HIV-infected cells to NK lysis and as a result play a key role in maintaining the HIV reservoir by avoiding recognition by NK cells.

Characterization of occult hepatitis B virus from blood donors carrying genotype A2 or genotype D strains.

BACKGROUND/AIMS: Nucleic acid testing (NAT) for hepatitis B virus (HBV) DNA in blood donations identified occult HBV infection (OBI) as a potential threat to blood safety. METHODS: A collaborative study was undertaken to explore the molecular basis of OBIs prevalent in Europe in relation to clinical and serological data. RESULTS: Ninety-one percent of 77 donor samples of European origin HBV DNA positive but HBV surface antigen (HBsAg) negative were confirmed. Viral load ranged between unquantifiable and 5640 IU/mL (median 25 IU/mL). Fifty-two strains were genotyped (14 HBV(A2) and 38 HBV(D)). Compared to HBsAg+ samples, genotype D was significantly more frequent than genotype A2 in OBIs from Poland or Italy (P<0.04). Amino acid substitutions were concentrated in the immunologically active parts of the Pre-S/S proteins (P<0.0001) affecting both cellular CD8 T-cell epitopes and B-cell neutralizing Major Hydrophilic Region epitopes. Substitutions were more frequent in OBIs than in HBsAg+ strains of both genotype D (P<0.001) and A2 (P<0.01), in OBIs of genotype D than A2 in the 'a' region (P<0.001) but not cellular epitopes, and in anti-HBs+ than anti-HBs- OBIs (P<0.001). CONCLUSIONS: Results support the hypothesis that humoral and cellular immune pressure on the HBV envelope proteins are major mechanisms generating OBI.

A probable case of hepatitis B virus transfusion transmission revealed after a 13-month-long window period.

BACKGROUND: Transfusion-transmitted hepatitis B virus (HBV) infection in recipients with drug-related immunodeficiency is rarely described in endemic areas. Hepatitis B surface antigen (HBsAg)-negative infectious donor blood can be identified by sensitive nucleic acid testing (NAT). Two immunodeficient patients who received blood components from a single seronegative blood donor subsequently found to contain HBV DNA are described. MATERIALS AND METHODS: Multiple samples from the implicated donor and the two recipients were tested for HBV serologic and molecular markers. HBV genome fragments were amplified, sequenced, and phylogenetically analyzed. RESULTS: The implicated donation had low-level HBV DNA due to the donor being in the window period before the donor's seroconversion. Recipient 1 had been vaccinated to HBV and carried anti-HBs but remained negative for all other HBV markers until she developed acute hepatitis B (viral load 2.7 x 10(8) IU/mL and alanine aminotransferase [ALT] level 1744 IU/L) 13 months after transfusion of red cells. Identical HBV sequences from both donor and recipient provided evidence of transfusion-related infection. Recipient 2, who received platelets from the same donation while receiving major chemotherapy, remained uninfected. CONCLUSIONS: In unusual circumstances, HBV incubation time can be considerably prolonged. Both active and passive neutralizing antibodies to HBV likely delayed, but did not prevent, acute infection when the immune system was impaired. HBV NAT may have interdicted the infectious unit, although the donation viral load could not be quantified and odds of detection calculated.

Anti-HBs positive occult hepatitis B virus carrier blood infectious in two transfusion recipients.

BACKGROUND/AIMS: Occult hepatitis B infection (OBI) in blood donations is not considered infectious when anti-HBs is present. METHODS: Four months after transfusion of eight blood components during coronary arterial bypass surgery, a 59-year-old patient developed acute hepatitis B. A second 71-year-old patient transfused with a red cell concentrate (RCC) from one of these donations had early HBV infection 7 months post-transfusion. Samples were tested for HBV serological markers and HBV DNA was quantified and sequenced. RESULTS: One implicated donation contained anti-HBc, anti-HBs (12 IU/L) and 180 IU/ml of HBV DNA. Previous and subsequent samples contained 3-10 times lower viral load and slightly variable anti-HBs. Two previous donations did not cause HBV infection. Recipients of the FFP and RCC from the index donation were both HBV infected and carried genotype D strains with sequences identical to the donor strain. CONCLUSIONS: Despite anti-HBs, an OBI carrier transmitted HBV to two immunocompetent transfusion recipients.