Transmission of Hepatitis B and D Viruses in an African Rural Community.

According to the World Health Organization (WHO), an estimated 257 million people worldwide are chronically infected with hepatitis B virus (HBV), with approximately 15 million of them being coinfected with hepatitis D virus (HDV). To investigate the prevalence and transmission of HBV and HDV within the general population of a rural village in Cameroon, we analyzed serum samples from most (401/448) of the villagers. HBV surface antigen (HBsAg) was detected in 54 (13.5%) of the 401 samples, with 15% of them also containing anti-HDV antibodies. Although Cameroon has integrated HBV vaccination into their Expanded Program on Immunization for newborns in 2005, an HBsAg carriage rate of 5% was found in children below the age of 5 years. Of the 54 HBsAg-positive samples, 49 HBV pre-S/S sequences (7 genotype A and 42 genotype E sequences) could be amplified by PCR. In spite of the extreme geographical restriction in the recruitment of study participants, a remarkable genetic diversity within HBV genotypes was observed. Phylogenetic analysis of the sequences obtained from PCR products combined with demographic information revealed that the presence of some genetic variants was restricted to members of one household, indicative of intrafamilial transmission, which appears to take place at least in part perinatally from mother to child. Other genetic variants were more widely distributed, reflecting horizontal interhousehold transmission. Data for two households with more than one HBV-HDV-coinfected individual indicate that the two viruses are not necessarily transmitted together, as family members with identical HBV sequences had different HDV statuses. IMPORTANCE This study revealed that the prevalence of HBV and HDV in a rural area of Cameroon is extremely high, underlining the pressing need for the improvement of control strategies. Systematic serological and phylogenetic analyses of HBV sequences turned out to be useful tools to identify networks of virus transmission within and between households. The high HBsAg carriage rate found among children demonstrates that implementation of the HBV birth dose vaccine and improvement of vaccine coverage will be key elements in preventing both HBV and HDV infections. In addition, the high HBsAg carriage rate in adolescents and adults emphasizes the need for identification of chronically infected individuals and linkage to WHO-recommended treatment to prevent progression to liver cirrhosis and hepatocellular carcinoma.

Development of Dengue Virus Serotype-Specific NS1 Capture Assays for the Rapid and Highly Sensitive Identification of the Infecting Serotype in Human Sera.

Dengue fever can be caused by one of four distinct dengue virus (DENV) serotypes that cocirculate in many parts of the world. Point of care serotype-specific nonstructural protein-1 (NS1) capture assays for the rapid serotyping of DENV in human sera would greatly support epidemiological surveillance and potentially also prognosis in individual patients. To ensure both serotype specificity and broad coverage of variants within serotypes, we have applied an innovative approach for the generation and selection of serotype-specific anti-NS1 mAbs. To elicit mAbs against conformational epitopes, NMRI mice were immunized with living HEK 293 transfectants expressing the native target Ags in multiple display on the cell surface. For each serotype, three different NS1 sequence variants were sequentially used for immunization of mice, hybridoma selection, and capture assay development, respectively. Selection of optimal combinations of capturing and detecting mAbs yielded highly sensitive and specific NS1 serotyping ELISAs (st-ELISAs) for the four serotypes. st-ELISA testing of 41 dengue patient sera showed a 100% concordance with the serotype determined by serotype-specific reverse transcriptase real-time quantitative PCR. The respective NS1 variants could be detected for ∼10 d after the onset of illness. Ab-dependent enhancement of DENV infections may be associated with a specific range of pre-existing anti-DENV serological Ab titers. Testing of patient sera with the developed st-ELISAs will not only be useful for epidemiological studies and surveillance, but it may also help to develop and validate assays that can distinguish protective versus enhancing Ab responses for risk assessment for the development of severe dengue disease in individual patients.

Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization.

BACKGROUND: Due to their rising incidence and progressive geographical spread, infections with mosquito-borne viruses, such as dengue (DENV), chikungunya and zika virus, have developed into major public health challenges. Since all of these viruses may cause similar symptoms and can occur in concurrent epidemics, tools for their differential diagnosis and epidemiological monitoring are of urgent need. RESULTS: Here we report the application of a novel strategy to rapidly generate monoclonal antibodies (mAbs) against native viral antigens, exemplified for the DENV nonstructural glycoprotein 1 (NS1). The described system is based on the immunization of mice with transfected mammalian cells expressing the target antigens in multiple displays on their cell surface and thereby presenting them efficiently to the host immune system in their native conformation. By applying this cell-based approach to the DENV NS1 protein of serotypes 1 (D1NS1) and 4 (D4NS1), we were able to rapidly generate panels of DENV NS1 serotype cross-reactive, as well as D1NS1- and D4NS1 serotype-specific mAbs. Our data show that the generated mAbs were capable of recognizing the endogenous NS1 protein in DENV-containing biological samples. CONCLUSION: The use of this novel immunization strategy, allows for a fast and efficient generation of hybridoma cell lines, producing mAbs against native viral antigens. Envisaged applications of the mAbs include the development of test platforms enabling a differentiation of the DENV serotypes and high resolution immunotyping for epidemiological studies.

Limited Genetic Diversity of Hepatitis B Virus in the General Population of the Offin River Valley in Ghana.

Hepatitis B virus (HBV) infections account for approximately 780,000 deaths per year, most of which occur in the developing world. Co-infection with HBV and hepatitis delta virus (HDV) may lead to the most severe form of viral hepatitis. In Ghana, knowledge on the prevalence of HBV and HDV in the general population is scanty and the few genetic analyses of the prevailing HBV genotypes are dating back more than a decade. In the present study, 1,323 serum samples from individuals living in a rural area (Offin river valley) of Ghana were analyzed for the presence of the hepatitis B surface antigen (HBsAg). Positive sera were subsequently tested for the presence of anti-HDV antibodies. A total of 107 (8%) sera were HBsAg positive with an 8.4% prevalence of anti-HDV antibodies among the HBsAg positives. Phylogenetic analysis based on HBV pre-S/S sequences, attributed all 52 typable samples to genotype E. All belonged to serotype ayw4. While 19 sequences clustered with those from a number of African countries, the other 33 formed a separate cluster distinguished by an intergroup mean distance of 1.5% from the pan-African HBV/E cluster. Successful implementation of HBV vaccination in the region was reflected by the low HBsAg carrier rate of 1.8% among children ≤11 years.

Torque teno virus in fecal samples of patients with gastroenteritis: prevalence, genogroups distribution, and viral load.

Torque teno virus (TTV, genus Alphatorquevirus, family Anelloviridae) is a DNA virus, highly prevalent in populations from around the world. TTV isolates have been classified into five main phylogenetic groups (1-5) showing a large genetic distance between them. The presence of TTV has been detected in feces. However, whether all five TTV genogroups are excreted in feces and the frequency of these events are presently unknown. The presence of TTV DNA was assessed in feces from 135 Brazilian (0-90 years old) patients with gastroenteritis by using three PCR methods, including real-time PCR. One hundred twenty one (91.1%) samples were positive with at least one method. Using a genogroup-specific assay, it was shown that all genogroups were present. Thirty-seven (27.4%), 27 (20.0%), 57 (42.2%), 29 (21.5%), and 33 (24.4%) fecal samples contained TTV isolates belonging to genogroups 1-5, respectively. Coinfections with two, three, four, and five TTV genogroups were found in 23 (17.0%), 15 (11.1%), 7 (5.2%), and 7 (5.2%) fecal samples, respectively. Thus, 52 (38.5%) samples contained more than one TTV genogroup. Viral loads ranged from 2.6 to 6.5 log genome equivalents per gram of feces. However, only moderate variations of viral load were noted depending on genogroup and number of coinfecting TTV genogroups. These results show a high prevalence and a diversity of TTV isolates in feces.