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Despite the significant public health problems associated with hepatitis B virus (HBV) in sub-Saharan Africa, many countries in this region do not have systematic HBV surveillance or genetic information on HBV circulating locally. Here, we report on the genetic characterization of 772 HBV strains from Tanzania. Phylogenetic analysis of the S-gene sequences showed prevalence of HBV genotype A (HBV/A, n =671, 86.9 %), followed by genotypes D (HBV/D, n =95, 12.3 %) and E (HBV/E, n =6, 0.8 %). All HBV/A sequences were further classified into subtype A1, while the HBV/D sequences were assigned to a new cluster. Among the Tanzanian sequences, 84 % of HBV/A1 and 94 % of HBV/D were unique. The Tanzanian and global HBV/A1 sequences were compared and were completely intermixed in the phylogenetic tree, with the Tanzanian sequences frequently generating long terminal branches, indicating a long history of HBV/A1 infections in the country. The time to the most recent common ancestor was estimated to be 188 years ago [95 % highest posterior density (HPD): 132 to 265 years] for HBV/A1 and 127 years ago (95 % HPD: 79 to 192 years) for HBV/D. The Bayesian skyline plot showed that the number of transmissions 'exploded' exponentially between 1960-1970 for HBV/A1 and 1970-1990 for HBV/D, with the effective population of HBV/A1 having expanded twice as much as that of HBV/D. The data suggest that Tanzania is at least a part of the geographic origin of the HBV/A1 subtype. A recent increase in the transmission rate and significant HBV genetic diversity should be taken into consideration when devising public health interventions to control HBV infections in Tanzania.

Globally, hepatitis C Virus (HCV) infection is responsible for a large proportion of persons with liver disease, including cancer. The infection is highly prevalent in sub-Saharan Africa. West Africa was identified as a geographic origin of two HCV genotypes. However, little is known about the genetic composition of HCV populations in many countries of the region. Using conventional and next-generation sequencing (NGS), we identified and genetically characterized 65 HCV strains circulating among HCV-positive blood donors in Kumasi, Ghana. Phylogenetic analysis using consensus sequences derived from 3 genomic regions of the HCV genome, 5'-untranslated region, hypervariable region 1 (HVR1) and NS5B gene, consistently classified the HCV variants (n = 65) into genotypes 1 (HCV-1, 15%) and genotype 2 (HCV-2, 85%). The Ghanaian and West African HCV-2 NS5B sequences were found completely intermixed in the phylogenetic tree, indicating a substantial genetic heterogeneity of HCV-2 in Ghana. Analysis of HVR1 sequences from intra-host HCV variants obtained by NGS showed that three donors were infected with >1 HCV strain, including infections with 2 genotypes. Two other donors share an HCV strain, indicating HCV transmission between them. The HCV-2 strain sampled from one donor was replaced with another HCV-2 strain after only 2 months of observation, indicating rapid strain switching. Bayesian analysis estimated that the HCV-2 strains in Ghana were expanding since the 16th century. The blood donors in Kumasi, Ghana, are infected with a very heterogeneous HCV population of HCV-1 and HCV-2, with HCV-2 being prevalent. The detection of three cases of co- or super-infections and transmission linkage between 2 cases suggests frequent opportunities for HCV exposure among the blood donors and is consistent with the reported high HCV prevalence. The conditions for effective HCV-2 transmission existed for ~ 3-4 centuries, indicating a long epidemic history of HCV-2 in Ghana.

Hepatitis C virus (HCV) is classified into seven genotypes based on genetic diversity, and most genotypes have been found in Africa. Infections with HCV genotype 2 (HCV2) are most prevalent in West Africa and it was suggested that HCV2 originated in West Africa. To better understand the evolutionary epidemiology of HCV2 in Africa, we examined new NS5B sequences of HCV2 strains obtained from Côte d'Ivoire, Ghana and Nigeria sequenced at the Centers for Disease Control and Prevention with those available from West, North and Central Africa. Bayesian phylogeographic analysis using a discrete trait model showed that Ghana was the most likely geographical region for the origin of HCV2. Spread of HCV2 from Ghana did not appear to be through diffusion to adjacent countries along the coast. Rather, it was transmitted from Ghana to many distant countries in Africa, suggesting that certain routes of geographical dissemination were historically more efficient than mere proximity and that the HCV2 epidemic history in West Africa is extremely complex.

Unlabelled: The recent epidemic history of hepatitis B virus (HBV) infections in the United States is complex, as indicated by current disparity in HBV genotype distribution between acute and chronic hepatitis B cases and the rapid decline in hepatitis B incidence since the 1990s. We report temporal changes in the genetic composition of the HBV population using whole-genome sequences (n = 179) from acute hepatitis B cases (n = 1,206) identified through the Sentinel County Surveillance for Acute Hepatitis (1998 to 2006). HBV belonged mainly to subtypes A2 (75%) and D3 (18%), with times of their most recent common ancestors being 1979 and 1987, respectively. A2 underwent rapid population expansions in ca. 1995 and ca. 2002, coinciding with transient rises in acute hepatitis B notification rates among adults; D3 underwent expansion in ca. 1998. A2 strains from cases identified after 2002, compared to those before 2002, tended to cluster phylogenetically, indicating selective expansion of specific strains, and were significantly reduced in genetic diversity (P = 0.001) and frequency of drug resistance mutations (P = 0.001). The expansion of genetically close HBV A2 strains was associated with risk of infection among male homosexuals (P = 0.03). Incident HBV strains circulating in the United States were recent in origin and restricted in genetic diversity. Disparate transmission dynamics among phylogenetic lineages affected the genetic composition of HBV populations and their capacity to maintain drug resistance mutations. The tendency of selectively expanding HBV strains to be transmitted among male homosexuals highlights the need to improve hepatitis B vaccination coverage among at-risk adults., Importance: Hepatitis B virus (HBV) remains an important cause of acute and chronic liver disease globally and in the United States. Genetic analysis of HBV whole genomes from cases of acute hepatitis B identified from 1998 to 2006 in the United States showed dominance of genotype A2 (75%), followed by D3 (18%). Strains of both subtypes were recent in origin and underwent rapid population expansions from 1995 to 2000, indicating increase in transmission rate for certain HBV strains during a period of decline in the reported incidence of acute hepatitis B in the United States. HBV A2 strains from a particular cluster that experienced the most recent population expansion were more commonly detected among men who have sex with men. Vaccination needs to be stepped up to protect persons who remain at risk of HBV infection., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Chronic infection with hepatitis C virus (HCV) is a risk factor for liver diseases such as fibrosis, cirrhosis and hepatocellular carcinoma. HCV genetic heterogeneity was hypothesized to be associated with severity of liver disease. However, no reliable viral markers predicting disease severity have been identified. Here, we report the utility of sequences from 3 HCV 1b genomic regions, Core, NS3 and NS5b, to identify viral genetic markers associated with fast and slow rate of fibrosis progression (RFP) among patients with and without liver transplantation (n = 42)., Methods: A correlation-based feature selection (CFS) method was used to detect and identify RFP-relevant viral markers. Machine-learning techniques, linear projection (LP) and Bayesian Networks (BN), were used to assess and identify associations between the HCV sequences and RFP., Results: Both clustering of HCV sequences in LP graphs using physicochemical properties of nucleotides and BN analysis using polymorphic sites showed similarities among HCV variants sampled from patients with a similar RFP, while distinct HCV genetic properties were found associated with fast or slow RFP. Several RFP-relevant HCV sites were identified. Computational models parameterized using the identified sites accurately associated HCV strains with RFP in 70/30 split cross-validation (90-95% accuracy) and in validation tests (85-90% accuracy). Validation tests of the models constructed for patients with or without liver transplantation suggest that the RFP-relevant genetic markers identified in the HCV Core, NS3 and NS5b genomic regions may be useful for the prediction of RFP regardless of transplant status of patients., Conclusions: The apparent strong genetic association to RFP suggests that HCV genetic heterogeneity has a quantifiable effect on severity of liver disease, thus presenting opportunity for developing genetic assays for measuring virulence of HCV strains in clinical and public health settings.

The genetic characterization of hepatitis A virus (HAV) strains is commonly accomplished by sequencing subgenomic regions, such as the VP1/P2B junction. HAV genome is not extensively variable, thus presenting opportunity for sharing sequences of subgenomic regions among genetically unrelated isolates. The degree of misrepresentation of phylogenetic relationships by subgenomic regions is especially important for tracking transmissions. Here, we analyzed whole-genome (WG) sequences of 101 HAV strains identified from 4 major multi-state, food-borne outbreaks of hepatitis A in the Unites States and from 14 non-outbreak-related HAV strains that shared identical VP1/P2B sequences with the outbreak strains. Although HAV strains with an identical VP1/P2B sequence were specific to each outbreak, WG were different, with genetic diversity reaching 0.31% (mean 0.09%). Evaluation of different subgenomic regions did not identify any other section of the HAV genome that could accurately represent phylogenetic relationships observed using WG sequences. The identification of 2-3 dominant HAV strains in 3 out of 4 outbreaks indicates contamination of the implicated food items with a heterogeneous HAV population. However, analysis of intra-host HAV variants from eight patients involved in one outbreak showed that only a single sequence variant established infection in each patient. Four non-outbreak strains were found closely related to strains from 2 outbreaks, whereas ten were genetically different from the outbreak strains. Thus, accurate tracking of HAV strains can be accomplished using HAV WG sequences, while short subgenomic regions are useful for identification of transmissions only among cases with known epidemiological association.

Mass spectrometry (MS) has found numerous applications in life sciences. It has high accuracy, sensitivity and wide dynamic range in addition to medium- to high-throughput capabilities. These features make MS a superior platform for analysis of various biomolecules including proteins, lipids, nucleic acids and carbohydrates. Until recently, MS was applied for protein detection and characterization. During the last decade, however, MS has successfully been used for molecular diagnostics of microbial and viral infections with the most notable applications being identification of pathogens, genomic sequencing, mutation detection, DNA methylation analysis, tracking of transmissions, and characterization of genetic heterogeneity. These new developments vastly expand the MS application from experimental research to public health and clinical fields. Matching of molecular techniques with specific requirements of the major MS platforms has produced powerful technologies for molecular diagnostics, which will further benefit from coupling with computational tools for extracting clinical information from MS-derived data.

Objective: To determine whether improper high-level disinfection practices during endoscopy procedures resulted in bloodborne viral infection transmission., Design:  Retrospective cohort study., Setting:  Four Veterans Affairs medical centers (VAMCs)., Patients:  Veterans who underwent colonoscopy and laryngoscopy (ear, nose, and throat [ENT]) procedures from 2003 to 2009., Methods:  Patients were identified through electronic health record searches and serotested for human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV). Newly discovered case patients were linked to a potential source with known identical infection, whose procedure occurred no more than 1 day prior to the case patient's procedure. Viral genetic testing was performed for case/proximate pairs to determine relatedness., Results:  Of 10,737 veterans who underwent endoscopy at 4 VAMCs, 9,879 patients agreed to viral testing. Of these, 90 patients were newly diagnosed with 1 or more viral bloodborne pathogens (BBPs). There were no case/proximate pairings found for patients with either HIV or HBV; 24 HCV case/proximate pairings were found, of which 7 case patients and 8 proximate patients had sufficient viral load for further genetic testing. Only 2 of these cases, both of whom underwent laryngoscopy, and their 4 proximates agreed to further testing. None of the 4 remaining proximate patients who underwent colonoscopy agreed to further testing. Mean genetic distance between the 2 case patients and 4 proximate patients ranged from 13.5% to 19.1%., Conclusions:  Our investigation revealed that exposure to improperly reprocessed ENT endoscopes did not result in viral transmission in those patients who had viral genetic analysis performed. Any potential transmission of BBPs from colonoscopy remains unknown.

We investigated the molecular epidemiology and population dynamics of HCV infection among indigenes of two semi-isolated communities in North-Central Nigeria. Despite remoteness and isolation, ~15% of the population had serological or molecular markers of hepatitis C virus (HCV) infection. Phylogenetic analysis of the NS5b sequences obtained from 60 HCV-infected residents showed that HCV variants belonged to genotype 1 (n=51; 85%) and genotype 2 (n=9; 15%). All sequences were unique and intermixed in the phylogenetic tree with HCV sequences from people infected from other West African countries. The high-throughput 454 pyrosequencing of the HCV hypervariable region 1 and an empirical threshold error correction algorithm were used to evaluate intra-host heterogeneity of HCV strains of genotype 1 (n=43) and genotype 2 (n=6) from residents of the communities. Analysis revealed a rare detectable intermixing of HCV intra-host variants among residents. Identification of genetically close HCV variants among all known groups of relatives suggests a common intra-familial HCV transmission in the communities. Applying Bayesian coalescent analysis to the NS5b sequences, the most recent common ancestors for genotype 1 and 2 variants were estimated to have existed 675 and 286 years ago, respectively. Bayesian skyline plots suggest that HCV lineages of both genotypes identified in the Nigerian communities experienced epidemic growth for 200-300 years until the mid-20th century. The data suggest a massive introduction of numerous HCV variants to the communities during the 20th century in the background of a dynamic evolutionary history of the hepatitis C epidemic in Nigeria over the past three centuries.

Genomes of hepatitis E virus (HEV), rubivirus and cutthroat virus (CTV) contain a region of high proline density and low amino acid (aa) complexity, named the polyproline region (PPR). In HEV genotypes 1, 3 and 4, it is the only region within the non-structural open reading frame (ORF1) with positive selection (4-10 codons with dN/dS>1). This region has the highest density of sites with homoplasy values >0.5. Genotypes 3 and 4 show ∼3-fold increase in homoplastic density (HD) in the PPR compared to any other region in ORF1, genotype 1 does not exhibit significant HD (p<0.0001). PPR sequence divergence was found to be 2-fold greater for HEV genotypes 3 and 4 than for genotype 1. The data suggest the PPR plays an important role in host-range adaptation. Although the PPR appears to be hypervariable and homoplastic, it retains as much phylogenetic signal as any other similar sized region in the ORF1, indicating that convergent evolution operates within the major HEV phylogenetic lineages. Analyses of sequence-based secondary structure and the tertiary structure identify PPR as an intrinsically disordered region (IDR), implicating its role in regulation of replication. The identified propensity for the disorder-to-order state transitions indicates the PPR is involved in protein-protein interactions. Furthermore, the PPR of all four HEV genotypes contains seven putative linear binding motifs for ligands involved in the regulation of a wide number of cellular signaling processes. Structure-based analysis of possible molecular functions of these motifs showed the PPR is prone to bind a wide variety of ligands. Collectively, these data suggest a role for the PPR in HEV adaptation. Particularly as an IDR, the PPR likely contributes to fine tuning of viral replication through protein-protein interactions and should be considered as a target for development of novel anti-viral drugs.

Background: A high prevalence of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections have been reported among persons with severe mental illness. In October, 2009, the Cook County Department of Public Health (CCDPH) initiated an investigation following notification of a cluster of HBV infections among mentally ill residents at a long term care facility (LTCF)., Methods: LTCF staff were interviewed and resident medical records were reviewed. Residents were offered testing for HBV, HCV, and HIV. Serum specimens from residents diagnosed with HBV or HIV infection were sent to the Centers for Disease Control and Prevention (CDC) for analysis., Results: Eleven newly diagnosed HBV infections were identified among mentally ill residents at the LTCF. Of these 11 infections, 4 serum specimens were available for complete HBV genome sequencing; all 4 genomes were found to be closely related. Four newly diagnosed HIV infections were identified within this same population. Upon molecular analysis, 2 of 4 HIV sequences from these new infections were found to be nearly identical and formed a tight phylogenetic cluster., Conclusions: HBV and HIV transmission was identified among mentally ill residents of this LTCF. Continued efforts are needed to prevent bloodborne pathogen transmission among mentally ill residents in LTCFs.

The intrahost evolution of hepatitis C virus (HCV) holds keys to understanding mechanisms responsible for the establishment of chronic infections and to development of a vaccine and therapeutics. In this study, intrahost variants of two variable HCV genomic regions, HVR1 and NS5A, were sequenced from four treatment-naïve chronically infected patients who were followed up from the acute stage of infection for 9 to 18 years. Median-joining network analysis indicated that the majority of the HCV intrahost variants were observed only at certain time points, but some variants were detectable at more than one time point. In all patients, these variants were found organized into communities or subpopulations. We hypothesize that HCV intrahost evolution is defined by two processes: incremental changes within communities through random mutation and alternations between coexisting communities. The HCV population was observed to incrementally evolve within a single community during approximately the first 3 years of infection, followed by dispersion into several subpopulations. Two patients demonstrated this pattern of dispersion for the rest of the observation period, while HCV variants in the other two patients converged into another single subpopulation after ∼9 to 12 years of dispersion. The final subpopulation in these two patients was under purifying selection. Intrahost HCV evolution in all four patients was characterized by a consistent increase in negative selection over time, suggesting the increasing HCV adaptation to the host late in infection. The data suggest specific staging of HCV intrahost evolution.

Genetic analysis of hepatitis B virus (HBV) frequently involves study of intra-host variants, identification of which is commonly achieved using short regions of the HBV genome. However, the use of short sequences significantly limits evaluation of genetic relatedness among HBV strains. Although analysis of HBV complete genomes using genetic cloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approach to whole genome (WG) HBV quasispecies analysis based on end-point, limiting-dilution real-time PCR (EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispecies from serum samples of patients (n = 38) infected with HBV genotypes A, B, C, D, E and G. Phylogenetic analysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub-populations of the virus. A critical observation was that HBV-WG consensus sequences obtained by direct sequencing of PCR fragments without EPLD are genetically close, but not always identical to the major HBV variants in the intra-host population, thus indicating that consensus sequences should be judiciously used in genetic analysis. Sequence-based studies of HBV WG quasispecies should afford a more accurate assessment of HBV evolution in various clinical and epidemiological settings.

Background: Hepatitis E virus (HEV) is an enterically transmitted hepatropic virus. It segregates as four genotypes. All genotypes infect humans while only genotypes 3 and 4 also infect several animal species. It has been suggested that hepatitis E is zoonotic, but no study has analyzed the evolutionary history of HEV. We present here an analysis of the evolutionary history of HEV., Methods and Findings: The times to the most recent common ancestors for all four genotypes of HEV were calculated using BEAST to conduct a Bayesian analysis of HEV. The population dynamics for genotypes 1, 3 and 4 were analyzed using skyline plots. Bayesian analysis showed that the most recent common ancestor for modern HEV existed between 536 and 1344 years ago. The progenitor of HEV appears to have given rise to anthropotropic and enzootic forms of HEV, which evolved into genotypes 1 and 2 and genotypes 3 and 4, respectively. Population dynamics suggest that genotypes 1, 3 and 4 experienced a population expansion during the 20(th) century. Genotype 1 has increased in infected population size ∼30-35 years ago. Genotype 3 and 4 have experienced an increase in population size starting late in the 19(th) century until ca.1940-45, with genotype 3 having undergone additional rapid expansion until ca.1960. The effective population size for both genotype 3 and 4 rapidly declined to pre-expansion levels starting in ca.1990. Genotype 4 was further examined as Chinese and Japanese sequences, which exhibited different population dynamics, suggesting that this genotype experienced different evolutionary history in these two countries., Conclusions: HEV appears to have evolved through a series of steps, in which the ancestors of HEV may have adapted to a succession of animal hosts leading to humans. Analysis of the population dynamics of HEV suggests a substantial temporal variation in the rate of transmission among HEV genotypes in different geographic regions late in the 20(th) Century.

Background: In January 2008, 3 persons with acute hepatitis C who all underwent endoscopy at a single facility in Nevada were identified., Method: We reviewed clinical and laboratory data from initially detected cases of acute hepatitis C and reviewed infection control practices at the clinic where case patients underwent endoscopy. Persons who underwent procedures on days when the case patients underwent endoscopy were tested for hepatitis C virus (HCV) infection and other bloodborne pathogens. Quasispecies analysis determined the relatedness of HCV in persons infected., Results: In addition to the 3 initial cases, 5 additional cases of clinic-acquired HCV infection were identified from 2 procedure dates included in this initial field investigation. Quasispecies analysis revealed 2 distinct clusters of clinic-acquired HCV infections and a source patient related to each cluster, suggesting separate transmission events. Of 49 HCV-susceptible persons whose procedures followed that of the source patient on 25 July 2007, 1 (2%) was HCV infected. Among 38 HCV-susceptible persons whose procedures followed that of another source patient on 21 September 2007, 7 (18%) were HCV infected. Reuse of syringes on single patients in conjunction with use of single-use propofol vials for multiple patients was observed during normal clinic operations., Conclusions: Patient-to-patient transmission of HCV likely resulted from contamination of single-use medication vials that were used for multiple patients during anesthesia administration. The resulting public health notification of approximately 50,000 persons was the largest of its kind in United States health care. This investigation highlighted breaches in aseptic technique, deficiencies in oversight of outpatient settings, and difficulties in detecting and investigating such outbreaks.

Background: In 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 Findings: Despite remoteness and isolation, approximately 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 approximately 30-40 years., Conclusions: Our 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.

Motivation: Insufficient knowledge of general principles for accurate quantitative inference of biological properties from sequences is a major obstacle in the rationale design of proteins with predetermined activities. Due to this deficiency, protein engineering frequently relies on the use of computational approaches focused on the identification of quantitative structure-activity relationship (SAR) for each specific task. In the current article, a computational model was developed to define SAR for a major conformational antigenic epitope of the hepatitis C virus (HCV) non-structural protein 3 (NS3) in order to facilitate a rationale design of HCV antigens with improved diagnostically relevant properties., Results: We present an artificial neural network (ANN) model that connects changes in the antigenic properties and structure of HCV NS3 recombinant proteins representing all 6 HCV genotypes. The ANN performed quantitative predictions of the enzyme immunoassay (EIA) Signal/Cutoff (S/Co) profiles from sequence information alone with 89.8% accuracy. Amino acid positions and physicochemical factors strongly associated with the HCV NS3 antigenic properties were identified. The positions most significantly contributing to the model were mapped on the NS3 3D structure. The location of these positions validates the major associations found by the ANN model between antigenicity and structure of the HCV NS3 proteins., Availability: Matlab code is available at the following URL address: http://bio-ai.myeweb.net/box&#95;widget.html

Numerous hepatitis A outbreaks were linked to the consumption of raw molluscan shellfish in the United States between 1960 and 1989. However, there had been no major molluscan shellfish-associated hepatitis A outbreaks reported in the United States for more than a decade (1989 to 2004). Beginning in late August 2005, at least 10 clusters of hepatitis A illnesses, totaling 39 persons, occurred in four states among restaurant patrons who ate oysters. Epidemiologic data indicated that oysters were the source of the outbreak. Traceback information showed that the implicated oysters were harvested from specific Gulf Coast areas. A voluntary recall of oysters was initiated in September. Hepatitis A virus (HAV) was detected in multiple 25-g portions in one of two recalled samples, indicating that as many as 1 of every 15 oysters from this source was contaminated. Comparing 315 nucleotides within the HAV VPl-2B region, 100% homology was found among four amplicons recovered from a total of six independent experiments of the implicated oysters, and an identical HAV sequence was detected in sera from all 28 patient serum specimens tested. Ten percent heterogeneity over 315 nucleotides (31 variants) was observed between the outbreak strain (subgenotype 1A) and an HM-175 strain (subgenotype 1B) used in the laboratory where the oysters were processed. To our knowledge, this investigation is the first in the United States to identify an HAV-identical strain in persons with hepatitis A as well as in the food that was implicated as the source of their infections.

The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenic properties of the NS5 protein was studied by using recombinant proteins. A strong antigenic region was identified within the HCV NS5A protein at amino acids 2212 to 2313. Forty-five unique sequences encompassing this region were selected from GenBank and were compared to each other. The results of this analysis showed that the primary structure of this strong antigenic region is highly variable. Percent homology between different genotype sequences varied from 40.4 to 72.5%. Thirteen representative sequences from all six HCV genotypes were selected to design synthetic genes coding for this antigenic region. These genes were assembled by PCR from synthetic oligonucleotides and expressed in Escherichia coli as hybrid proteins with glutathione S-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n = 91) obtained from patients infected with HCV genotypes 1 through 6. All but two proteins immunoreacted with 62 to 93% of HCV anti-NS5-positive serum samples. Although a variable degree of genotype-specific antigenic reactivity was detected, only one protein demonstrated a noticeable preference to immunoreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immunoreacted with significantly different efficiency with HCV antibodies. Thus, sequence variability has a profound effect on the antigenic properties of the NS5A immunodominant regions. This observation should be taken into consideration in the development of diagnostic tests for the efficient detection of anti-HCV activity in serum specimens.

The neutralization epitope(s) of the hepatitis E virus (HEV) was studied by an in vitro neutralization assay using antibodies obtained by immunization of mice with 51 overlapping 30-mer synthetic peptides spanning the region 221-660 amino acids (aa) of the HEV open reading frame 2 encoded protein (pORF2) and 31 overlapping recombinant proteins of different sizes derived from the entire pORF2 of the HEV Burma strain. Antibodies against synthetic peptides and short recombinant proteins of approximately 100 aa did not neutralize HEV, suggesting the HEV neutralization epitope(s) is conformation-dependent. However, one recombinant protein of approximately 400 aa in length comprising the pORF2 sequence at position 274-660 aa as well as all truncated derivatives of this protein containing region 452-617 aa elicited antibodies, demonstrating HEV neutralizing activity. These findings establish for the first time that the minimal size fragment, designated pB166, that can efficiently model the neutralization epitope(s) is 166 aa in length and is located at position 452-617 aa of the HEV pORF2. Additionally, antibodies against pB166 were found to cross-neutralize three different HEV genotypes, suggesting that a common neutralization epitope(s) may exist within the different HEV genotypes. Thus, recombinant proteins constructed in this study may be considered as potential candidates for the development of an HEV subunit vaccine as well as for the development of highly sensitive and specific diagnostic tests., (Copyright 2001 Academic Press.)

A series of serum specimens obtained from two chimpanzees experimentally infected with hepatitis A virus (HAV), hepatitis C virus, and hepatitis G/GB-C virus were tested for TT virus (TTV) by polymerase chain reaction (PCR). All PCR fragments obtained from both animals were directly sequenced, and the nucleotide sequences were compared to each other and to all known TTV sequences. This comparison showed that both animals were infected simultaneously with four new TTV variants designated A, M1, M2, and M3. One chimpanzee was found to be infected with TTV only after HAV inoculation, whereas the other animal was infected with TTV before any experimental procedure was performed. A set of PCR primers specific for these four new TTV variants was used to amplify TTV-like sequences from nine naive chimpanzees. None of these animals was infected with the prototype TTV variant. Two of these animals, however, were infected with one of the new TTV variants, while one animal was infected with an additional new TTV variant designated T. Among 99 hepatitis patients, 29 were found to be infected with the prototype TTV variant. None of these human specimens was found to be positive by PCR specific for TTV variants A, M1, M2, and M3. Similarly, not a single specimen from a smaller subset of human serum samples was found to be positive for the TTV variant T. Phylogenetic analysis performed on all known TTV sequences demonstrated that TTV can be classified into 13 different, yet closely related TTV species, designated as TTV-I for the prototype variant through TTV-XIII. The new variants M1 and M2 were classified as two different genotypes of TTV-VI, variant M3 was classified as TTV-VII, variant A was classified as TTV-VIII, and variant T was classified as TTV-IX. Thus, the data obtained in this study suggest that TTV represents a large swarm of TTV-like species, some of which have not been detected in humans and circulate predominantly among chimpanzees., (Copyright 2000 Academic Press.)

TT virus (TTV) is a recently discovered infectious agent originally obtained from transfusion-related hepatitis. However, the causative link between the TTV infection and liver disease remains uncertain. Recent studies demonstrated that genome sequences of different TTV strains are significantly divergent. To assess genetic heterogeneity of the TTV genome in more detail, a sequence analysis of PCR fragments (271 bp) amplified from open reading frame 1 (ORF1) was performed. PCR fragments were amplified from 5 to 40% of serum specimens obtained from patients with different forms of hepatitis who reside in different countries (e.g., China, Egypt, Vietnam, and the United States) and from normal human specimens obtained from U.S. residents. A total of 170 PCR fragments were sequenced and compared to sequences derived from the corresponding TTV genome region deposited in GenBank. Genotypes 2 and 3 were found to be significantly more genetically related than any other TTV genotype. Moreover, three sequences were shown to be almost equally related to both genotypes 2 and 3. These observations suggest a merger of genotypes 2 and 3 into one genotype, 2/3. Additionally, five new groups of TTV sequences were identified. One group represents a new genotype, whereas the other four groups were shown to be more evolutionary distant from all known TTV sequences. The evolutionary distances between these four groups were also shown to be greater than between TTV genotypes. The phylogenetic analysis suggested that these four new genetic groups represent closely related yet different viral species. Thus, TTV exists as a "swarm" of at least five closely related but different viruses. These observations suggest a high degree of genetic complexity within the TTV population. The finding of the additional TTV-related species should be taken into consideration when the association between TTV infections and human diseases of unknown etiology is studied.

The antigenic composition of the hepatitis E virus (HEV) protein encoded by open reading frame 2 (ORF2) was determined by using synthetic peptides. Three sets of overlapping 18-, 25-, and 30-mer peptides, with each set spanning the entire ORF2 protein of the HEV Burma strain, were synthesized. All synthetic peptides were tested by enzyme immunoassay against a panel of 32 anti-HEV-positive serum specimens obtained from acutely HEV-infected persons. Six antigenic domains within the ORF2 protein were identified. Domains 1 and 6 located at the N and C termini of the ORF2 protein, respectively, contain strong immunoglobulin G (IgG) and IgM antigenic epitopes that can be efficiently modeled with peptides of different sizes. In contrast, antigenic epitopes identified within the two central domains (3 and 4) were modeled more efficiently with 30-mer peptides than with either 18- or 25-mers. Domain 2 located at amino acids (aa) 143 to 222 was modeled best with 25-mer peptides. A few 30-mer synthetic peptides derived from domain 5 identified at aa 490 to 579 demonstrated strong IgM antigenic reactivity. Several 30-mer synthetic peptides derived from domains 1, 4, and 6 immunoreacted with IgG or IgM with more than 70% of anti-HEV-positive serum specimens. Thus, the results of this study demonstrate the existence of six diagnostically relevant antigenic domains within the HEV ORF2 protein.

Forty-two antigenic domains were identified across the hepatitis A virus (HAV) polyprotein by using a set of 237 overlapping 20-mer synthetic peptides spanning the entire HAV polyprotein and a panel of serum samples from acutely HAV-infected patients. The term "antigenic domain" is used in this study to define a protein region spanned with consecutive overlapping immunoreactive peptides. Nineteen antigenic domains were found within the structural proteins, and 22 were found within the nonstructural proteins, with 1 domain spanning the junction of VP1 and P2A proteins. Five of these domains were considered immunodominant, as judged by both the breadth and the strength of their immunoreactivity. One domain is located within the VP2 protein at position 57-90 aa. A second domain, located at position 767-842 aa, contains the C-terminal part of the VP1 protein and the entire P2A protein. A third domain, located at position 1403-1456 aa, comprises the C-terminal part of the P2C protein and the N-terminal half of the P3A protein. The fourth domain, located at position 1500-1519 aa, includes almost the entire P3B, and the last domain, located at position 1719-1764 aa, contains the C-terminal region of the P3C protein and the N-terminal region of the P3D protein. It is interesting to note that four of the five most immunoreactive domains are derived from small HAV proteins and/or encompass protein cleavage sites separating different HAV proteins. The HAV-specific immunoreactivity of each antigenically reactive peptide was confirmed by using seven HAV seroconversion panels. Collectively, these data demonstrate that HAV structural and nonstructural proteins contain antigenic epitopes that can be efficiently modeled with short synthetic peptides.

The effect of sequence heterogeneity on the immunologic properties of two strong antigenic regions of the hepatitis C virus (HCV) NS4 protein was studied by using a set of 443 overlapping 20-mer synthetic peptides. One antigenic region comprising the cleavage site between NS4a and NS4b (region 5-1-1) was modeled with peptides derived from 73 different known sequences, representing HCV genotypes 1-6. The other antigenic region, designated region 59 and located at the C-terminus of the NS4b protein, was modeled with peptides from 7 known sequences representing genotypes 1-3. All peptides were tested for antigenic reactivity by enzyme immunoassay with a panel of anti-HCV-positive serum specimens representing genotypes 1-5. The data demonstrated that immunoreactive peptides fell into two groups. One group, represented by N-terminal peptides, demonstrated genotype-independent immunoreactivity; the other group, from the central part of region 5-1-1, showed strict genotype specificity. Nineteen peptides from the genotype-independent group strongly immunoreacted with a wide range of serum samples containing antibodies to all 5 HCV genotypes. Twenty-five peptides from the genotype-specific group were found to strongly react with serum containing antibodies only to the genotype from which the peptides were derived. Similar to the N-terminal part of region 5-1-1, peptides derived from region 59 did not show genotype-specific immunoreactivity. Some peptides derived from the central part of region 59 showed very strong and broad antigenic reactivity. Thus, after examining two antigenic regions of the NS4 protein, we identified short sequences that can be used for the efficient detection of either genotype-independent or genotype-specific HCV antibodies.

Two sets of primers derived from the 5'-terminal region and the NS5 region of the hepatitis G virus (HGV) genome were used to amplify PCR fragments from serum specimens obtained from different parts of the world. All PCR fragments from the 5'-terminal region (5'-PCR, n = 56) and from the NS5 region (NS5-PCR, n = 85) were sequenced and compared to corresponding published HGV sequences. The range of nucleotide sequence similarity varied from 74 and 78% to 100% for 5'-PCR and NS5-PCR fragments, respectively. Additionally, five overlapping PCR fragments comprising an approximately 2.0-kb structural region of the HGV genome were sequenced from each of five sera obtained from three United States residents. These sequences were compared to 20 published sequences comprising the same region of the HGV genome. Nucleotide and deduced amino acid sequences obtained from different individuals were homologous from 82.9 to 93. 6% and from 90.4 to 99.0%, respectively. Sequences obtained from follow-up specimens were almost identical. Comparative analysis of deduced amino acid sequences of the HGV structural proteins and hepatitis C virus (HCV) structural proteins combined with an analysis of predicted secondary structures and hydrophobic profiles allowed prediction of processing sites within the HGV structural proteins. A phylogenetic sequence analysis performed on the 2.0-kb structural region supports the existence of three previously identified HGV genetic groups. However, phylogenetic analysis performed on only small DNA fragments yielded inconsistent genetic grouping and failed to confirm the existence of genetic groups. Thus, in contrast to HCV where almost any region can be used for genotyping, only large or carefully selected genome fragments can be used to identify consistent HGV genetic groups., (Copyright 1999 Academic Press.)

A recently developed polymerase chain reaction (PCR)-based cell culture neutralization assay was used to investigate cross-neutralization of known hepatitis E virus (HEV) strains obtained from various HEV-endemic regions of the world with different anti-HEV-positive serum samples. Serum specimens obtained from cynomolgus macaques experimentally infected with strains from Burma, Mexico, or Pakistan cross-neutralized the infectivity of each strain as well as an isolate from Morocco. Serum samples obtained either from infected patients who reside in HEV-endemic regions of the world or from U.S. residents who became infected while traveling to such regions also neutralized all four strains. In contrast, antibodies obtained from rabbits immunized with full-length Burma strain ORF2 protein neutralized only the Burma and Pakistan strains, not the Mexico or Morocco strains. In addition, antibodies obtained from guinea pigs immunized with an N-terminal truncated Burma strain ORF2 protein neutralized each strain except the Morocco strain. These data strongly suggest that antibodies elicited during an HEV infection demonstrate broad HEV neutralizing activity, whereas antibodies elicited after immunization with recombinant Burma ORF2 protein demonstrate a more limited ability to neutralize various HEV strains obtained from different regions of the world endemic for the disease., (Copyright 1998 Academic Press.)

Recently, a new virus related to flaviviruses, the hepatitis G virus (HGV), or GBV-C virus, was discovered as a putative blood-borne human pathogen. HGV RNA (NS5 region) was amplified by reverse transcription-nested PCR in the sera of 6 of 64 (9%) hemodialysis patients; 2 of 80 (2.5%) West Yukpa Amerindians, a population with a high rate of HBV infection but negative for HCV infection; and 1 patient with an acute episode of non-A, non-B, non-C hepatitis (NABCH). The patterns of single-strand conformation polymorphism of the amplified products were unique among different specimens and similar on follow-up for hemodialysis patients. All patients tested remained HGV RNA positive 1 and 2 years later, without major sequence variation, except for the NABCH patient, for whom a double infection and an apparent clearance of the original dominant variant was observed after 2 years. The sequences of the NS5 amplified products demonstrated 85 to 90% identity with other reported HGV sequences.

Antibody reactivities to hepatitis C virus (HCV) antigens and to synthetic peptides derived from different parts of the HCV genome (core, NS4, and NS5) were evaluated in HCV-infected hemodialysis patients. In the RIBA 3 assay, NS5 was significantly less recognizable by sera of hemodialysis patients compared to other HCV-infected subjects. Among hemodialysis patients, those coinfected with hepatitis B virus (HBV) (positive for hepatitis B surface antigen [HBsAg+]) showed a reduction in reactivity to C33 and C100. Sera of only 23% of the hemodialysis patients (37 of 161) reacted with more than three of eight peptides tested, significantly fewer than the 60% (12 of 20) of the sera of other HCV-infected patients tested (P = 0.001). This immunosuppression was also manifested by a reduced frequency of recognition of additional peptides on follow-up. An even more reduced reactivity was observed among the HBV-coinfected patients (HBsAg+). The low-responder hemodialysis patients were not infected with any particular genotype of HCV, and the same HCV genotypes observed in the whole group of hemodialysis patients (1a, 1b, 2a, and 3a) were found circulating in the low-responder group. Even in this low-responder population, the good performance of two peptides (peptide 716, corresponding to a portion of the core, and peptide 59, corresponding to a portion of NS4) corroborates the immunodominance of the conserved epitopes within these peptides.

Nine sets of nested PCR primers from a 2.6-kb region of the hepatitis G virus (HGV) genome at nucleotide positions 5829 to 8421 were designed and used to analyze serum specimens obtained from patients with community-acquired non-A, non-B hepatitis who were HGV RNA positive. One set of primers was found to be most efficient in detecting HGV and was subsequently used to test 162 HCV-positive and 11 HCV-negative plasma units obtained from individual paid donors. HGV RNA was detected in 30 (17.3%) plasma units, 2 of which were found among the 11 HCV-negative specimens. A complete set of nine PCR fragments was obtained from two patients with community-acquired acute non-A, non-B hepatitis and from four paid donors. All PCR fragments were sequenced and were shown to have a nucleotide similarity of 85.9 to 92.3% and a derived amino acid similarity of 96.0 to 99.0%. The majority of nucleotide changes occurred in the third position of codons. The HGV nucleotide and protein sequences obtained in this study were compared with HCV sequences. Based on this analysis the 2.6-kb fragment was predicted to encode the C-terminal part of the putative NS4b, the entire NS5a, and almost the complete NS5b proteins. Putative protease cleavage sites separating these proteins were also predicted. In serial specimens obtained from the two HGV-infected patients, no significant variations were found in the HGV nucleotide and derived amino acid sequences over time. The HGV sequences obtained from one patient showed no changes over 6 months, whereas more than 99.0% homology was observed for sequences from the second patient over 2.5 years. Heterogeneity analysis performed on 10 sequences obtained in this study and corresponding regions from 6 known full-size sequences of the HGV genomes demonstrated notable discrete heterogeneity consistent with the existence of HGV genetic groups or types.

The role of hepatitis G virus (HGV) infection in acute non-A-E hepatitis was investigated in adults with viral hepatitis. HGV RNA was present in 1 of 28 patients with non-A-E hepatitis but 9 of 22 with hepatitis C (P < .003). HGV RNA-positive patients (HGV-infected and HGV-hepatitis C virus [HCV]-coinfected) developed light-to-moderate jaundice. Clinical and biochemical features of HGV-positive and HCV-positive patients and patients with non-A, non-G hepatitis were similar. Three patients with HGV-HCV coinfection, tested within 18 months after disease onset, have remained HGV RNA-positive but have become HCV RNA-negative. Only 1 non-A-E hepatitis patient was confirmed as being infected with HGV alone, suggesting that HGV is not the main etiologic agent of non-A-E hepatitis. Although HGV RNA was significantly associated with hepatitis C, patients with mixed HCV-HGV infections did not demonstrate a more severe course of disease than did patients with HCV infection.

Hepatitis delta virus (HDV) is known to express a protein termed the small delta antigen, a structural protein which is also essential for genome replication. During replication, posttranscriptional RNA editing specifically modifies some of the HDV RNA, leading to the production of an elongated form of the delta antigen, the large form, which is essential for virus assembly. The present study showed that yet another form of HDV protein is expressed during genome replication. This novel form is not produced in all infected cells, but it arises during replication in transfected cells and in infected woodchucks, and as was previously reported, patients infected with HDV do make antibodies directed against it. These findings are an indicator of the complexity of gene expression during HDV infection and replication.

A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera. The data obtained strongly indicate a diagnostic potential for the mosaic protein.

A synthetic gene encoding the hepatitis C virus (HCV) nucleocapsid protein was constructed and expressed in E. coli. To synthesize this gene, we developed a new method that results in the enzymatic synthesis of long polydeoxyribonucleotides from oligodeoxyribonucleotides. The method, designated as the 'Exchangeable Template Reaction' (ETR), uses oligonucleotides as templates for DNA polymerase. A special mechanism was designed to exchange the templates during the polymerase reaction. The mechanism relies on the formation of a single-stranded 3'-protrusion at the 'growing point' of the elongating DNA such that it can be subsequently annealed, in a sequence-specific manner, with the next synthetic oligonucleotide. When annealed to the 3'-protrusion, the added oligonucleotide becomes a template for DNA polymerase, and the protruding 3'-end of the double-stranded DNA is used as the primer. The HCV nucleocapsid gene was assembled with DNA ligase from three fragments synthesized by ETR. The data verify that this method is efficient. The main advantage of ETR is the ability to combine more than two oligonucleotides in one tube together with polymerase and an enzymatic activity that produces a 3'-protrusion (e.g., BstXI) rather than the sequential addition of each component. The data demonstrate that as many as five oligonucleotides can be used simultaneously, resulting in a synthesized DNA fragment of designed sequence. The synthetic gene expressed in E. coli produced a 27 kDa protein that specifically interacted with antibodies from sera obtained from HCV-infected individuals.