SUMMARY Current serologic tests provide the foundation for diagnosis of hepatitis A and hepatitis A virus (HAV) infection. Recent advances in methods to identify and characterize nucleic acid markers of viral infections have provided the foundation for the field of molecular epidemiology and increased our knowledge of the molecular biology and epidemiology of HAV. Although HAV is primarily shed in feces, there is a strong viremic phase during infection which has allowed easy access to virus isolates and the use of molecular markers to determine their genetic relatedness. Molecular epidemiologic studies have provided new information on the types and extent of HAV infection and transmission in the United States. In addition, these new diagnostic methods have provided tools for the rapid detection of food-borne HAV transmission and identification of the potential source of the food contamination.

Green onions that were apparently contaminated before arrival at the restaurant caused this unusually large foodborne outbreak of hepatitis A. The inclusion of contaminated green onions in large batches that were served to all customers contributed to the size of the outbreak.

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Hepatitis C is a major public health problem in the United States and worldwide. Outbreaks of hepatitis C virus (HCV) infections are associated with unsafe injection practices, drug diversion, and other exposures to blood, being difficult to detect and investigate. Here, we developed and validated a simple approach for molecular detection of HCV transmissions in outbreak settings. We obtained sequences from the HCV hypervariable region 1 (HVR1) using End-Point Limiting-Dilution (EPLD) from 127 cases involved in 32 epidemiologically defined HCV outbreaks and 193 individuals with unrelated HCV strains. We compared several types of genetic distances and calculated a threshold using minimal Hamming distances that identifies transmission clusters in all tested outbreaks with 100% accuracy. The approach was also validated on sequences from 239 individuals obtained using next-generation sequencing, showing the same accuracy as EPLD. In average, nucleotide diversity of the intra-host population was 6.2-times greater in the source than in any incident case, allowing the correct detection of transmission direction in 8 outbreaks for which source cases were known. A simple and accurate distance-based approach for detecting HCV transmissions developed here streamlines molecular investigation of outbreaks, thus improving the public health capacity for rapid and effective control of hepatitis C.

BackgroundIn Nigeria, hepatitis B virus (HBV) infection has reached hyperendemic levels and its nature and origin have been described as a puzzle. In this study, we investigated the molecular epidemiology and epidemic history of HBV infection in two semi-isolated rural communities in North/Central Nigeria. It was expected that only a few, if any, HBV strains could have been introduced and effectively transmitted among these residents, reflecting limited contacts of these communities with the general population in the country.Methods and FindingsDespite remoteness and isolation, ∼11% of the entire population in these communities was HBV-DNA seropositive. Analyses of the S-gene sequences obtained from 55 HBV-seropositive individuals showed the circulation of 37 distinct HBV variants. These HBV isolates belong predominantly to genotype E (HBV/E) (n = 53, 96.4%), with only 2 classified as sub-genotype A3 (HBV/A3). Phylogenetic analysis showed extensive intermixing between HBV/E variants identified in these communities and different countries in Africa. Quasispecies analysis of 22 HBV/E strains using end-point limiting-dilution real-time PCR, sequencing and median joining networks showed extensive intra-host heterogeneity and inter-host variant sharing. To investigate events that resulted in such remarkable HBV/E diversity, HBV full-size genome sequences were obtained from 47 HBV/E infected persons and P gene was subjected to Bayesian coalescent analysis. The time to the most recent common ancestor (tMRCA) for these HBV/E variants was estimated to be year 1952 (95% highest posterior density (95% HPD): 1927–1970). Using additional HBV/E sequences from other African countries, the tMRCA was estimated to be year 1948 (95% HPD: 1924–1966), indicating that HBV/E in these remote communities has a similar time of origin with multiple HBV/E variants broadly circulating in West/Central Africa. Phylogenetic analysis and statistical neutrality tests suggested rapid HBV/E population expansion. Additionally, skyline plot analysis showed an increase in the size of the HBV/E-infected population over the last ∼30–40 years.ConclusionsOur data suggest a massive introduction and relatively recent HBV/E expansion in the human population in Africa. Collectively, these data show a significant shift in the HBV/E epidemic dynamics in Africa over the last century.

Low rates of hepatitis A in both groups indicate that hepatitis A vaccine and immune globulin provided good protection after exposure. Although the study's prespecified criterion for noninferiority was met, the slightly higher rates of hepatitis A among vaccine recipients may indicate a true modest difference in efficacy and might be clinically meaningful in some settings. Vaccine has other advantages, including long-term protection, and it may be a reasonable alternative to immune globulin for postexposure prophylaxis in many situations. (ClinicalTrials.gov number, NCT00139139 [ClinicalTrials.gov].).

Although dengue virus (DV) enters through skin while mosquitoes feed, early contacts remain unexplored regarding the cutaneous viral fate and in situ immune responses. We addressed this by exposing healthy, non-cadaveric, freshly obtained human skin explants to a human DV2 isolate. We demonstrated negative-strand DV-RNA and non-structural protein-1, both suggestive of viral replication in skin. Although control, mock-infected and DV-infected explants showed less (MHC-CII(+)/CD1a(+)/Langerin+) Langerhans cells, deranged morphology and decreased frequency were more apparent in DV-infected explants. Whereas DV+ cells were infrequent in epidermis and completely absent in dermis, some areas of basal epidermis were clearly DV+, presumably keratinocytes, cells where TUNEL positivity revealed apoptosis. Unlike fresh, control and mock-infected skin, DV-infected explants expressed CD80 and CD83, indicative of dendritic cell (DC) activation and maturation, respectively. However, sequential sections indicated that these cells were not DV+, suggesting that activated/mature DCs capable of priming T cells, probably, were not infected. Alternatively, the occasionally infected epidermal DC might not have reached maturation. Interestingly, skin DV infection apparently uncouples the DC activation/maturation process from another crucial DC function, the subsequent migration into dermis. This was suggested, because upon cutaneous DV infection, the few emerging CD83+ (mature) DCs remained within the outer epidermis, while no dermal CD83+ DCs were observed. These paradoxical effects might represent unknown DV subversion strategies. This approach is relatively easy, quick (results in 48 h), economical for developing countries where dengue is re-emerging and advantageous to evaluate in situ viral biology, immunity and immunopathology and potential antiviral strategies.

BackgroundNext-generation sequencing allows the analysis of an unprecedented number of viral sequence variants from infected patients, presenting a novel opportunity for understanding virus evolution, drug resistance and immune escape. However, sequencing in bulk is error prone. Thus, the generated data require error identification and correction. Most error-correction methods to date are not optimized for amplicon analysis and assume that the error rate is randomly distributed. Recent quality assessment of amplicon sequences obtained using 454-sequencing showed that the error rate is strongly linked to the presence and size of homopolymers, position in the sequence and length of the amplicon. All these parameters are strongly sequence specific and should be incorporated into the calibration of error-correction algorithms designed for amplicon sequencing.ResultsIn this paper, we present two new efficient error correction algorithms optimized for viral amplicons: (i) k-mer-based error correction (KEC) and (ii) empirical frequency threshold (ET). Both were compared to a previously published clustering algorithm (SHORAH), in order to evaluate their relative performance on 24 experimental datasets obtained by 454-sequencing of amplicons with known sequences. All three algorithms show similar accuracy in finding true haplotypes. However, KEC and ET were significantly more efficient than SHORAH in removing false haplotypes and estimating the frequency of true ones.ConclusionsBoth algorithms, KEC and ET, are highly suitable for rapid recovery of error-free haplotypes obtained by 454-sequencing of amplicons from heterogeneous viruses.The implementations of the algorithms and data sets used for their testing are available at: http://alan.cs.gsu.edu/NGS/?q=content/pyrosequencing-error-correction-algorithm