Recent and occult hepatitis B virus infections among blood donors in the United States.

BACKGROUND: Characteristics of US blood donors with recent (RBI) or occult (OBI) hepatitis B virus (HBV) infection are not well defined. METHODS: Donors with RBI and OBI were identified by nucleic acid and serologic testing among 34.4 million donations during 2009-2015. Consenting donors were interviewed and their HBV S-gene sequenced. RESULTS: The overall rate of HBV-infected donors was 7.95 per 100,000; of these, 0.35 per 100,000 and 1.70 per 100,000 were RBI and OBI, respectively. RBI (n = 120) and OBI (n = 583) donors constituted 26% of all HBV-infected (n = 2735) donors. Detection of HBV DNA in 92% of OBI donors required individual donation nucleic acid testing. Donors with OBI compared to RBI were older (mean age, 48 vs 39 years; p < 0.0001) with lower median viral loads (9 vs. 529 IU/mL; p < 0.0001). A higher proportion of OBI than RBI donors were born or resided in an endemic country (39% vs. 5%; p = 0.0078). Seventy-seven percent of all RBI and OBI donors had multiple sex partners, an HBV-risk factor. Of 40 RBI and 10 OBI donors whose S gene was sequenced, 33 (83%) and 6 (60%), respectively, carried HBV subgenotype A2; 18 (55%) and 2 (33%), respectively, shared an identical sequence. Infection with 1 or more putative HBV-immune-escape mutants was identified in 5 (50%) of OBI but no RBI donors. CONCLUSION: RBI and OBI continue to be identified at low rates, confirming the importance of comprehensive HBV DNA screening of US blood donations. HBV-infected donors require referral for care and evaluation and contact tracing; their HBV strains may provide important information on emergent genotypes.

Molecular epidemiology of hepatitis B virus infection in Tanzania.

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.

High frequency of active HCV infection among seropositive cases in west Africa and evidence for multiple transmission pathways.

BACKGROUND: Sub-Saharan Africa (SSA) has one of the highest global hepatitis C virus (HCV) prevalence estimates. However, reports that suggest high rates of serologic false positives and low levels of viremia have led to uncertainty regarding the burden of active infection in this region. Additionally, little is known about the predominant transmission risk factors in SSA. METHODS: We prospectively recalled 363 past blood donors (180 who were rapid screen assay [RSA] positive and 183 who were RSA negative at time of donation) to identify the level of active infection and risk factors for infection at a teaching hospital in Kumasi, Ghana. Participants had repeat blood testing and were administered a questionnaire on risk factors. RESULTS: The frequency of HCV active infection ranged from 74.4% to 88% depending on the criteria used to define serologically positive cases. Individuals with active disease had biochemical evidence of liver inflammation and median viral loads of 5.7 log copies/mL. Individuals from the northern and upper regions of Ghana had greater risks of infection compared with participants from other areas. Additional risk factors included traditional circumcision, home birth, tribal scarring, and hepatitis B virus coinfection. CONCLUSIONS: Viremic infection was common among serologically confirmed cases. Attention to testing algorithms is needed in order to define the true HCV burden in SSA. These data also suggest that several transmission modes are likely contributing to the current HCV epidemic in Ghana and that the distribution of these practices may result in substantial regional variation in prevalence.

A re-evaluation of the origin of hepatitis C virus genotype 2 in West Africa.

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.

Recent population expansions of hepatitis B virus in the United States.

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.

Computational models of liver fibrosis progression for hepatitis C virus chronic infection.

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.

Intra-host diversity and evolution of hepatitis C virus endemic to Côte d'Ivoire.

Hepatitis C virus (HCV) infection presents an important, but underappreciated public health problem in Africa. In Côte d'Ivoire, very little is known about the molecular dynamics of HCV infection. Plasma samples (n = 608) from pregnant women collected in 1995 from Côte d'Ivoire were analyzed in this study. Only 18 specimens (∼3%) were found to be HCV PCR-positive. Phylogenetic analysis of the HCV NS5b sequences showed that the HCV variants belong to genotype 1 (HCV1) (n = 12, 67%) and genotype 2 (HCV2) (n = 6, 33%), with a maximum genetic diversity among HCV variants in each genotype being 20.7% and 24.0%, respectively. Although all HCV2 variants were genetically distant from each other, six HCV1 variants formed two tight sub-clusters belonging to HCV1a and HCV1b. Analysis of molecular variance (AMOVA) showed that the genetic structure of HCV isolates from West Africa with Côte d'Ivoire included were significantly different from Central African strains (P = 0.0001). Examination of intra-host viral populations using next-generation sequencing of the HCV HVR1 showed a significant variation in intra-host genetic diversity among infected individuals, with some strains composed of sub-populations as distant from each other as viral populations from different hosts. Collectively, the results indicate a complex HCV evolution in Côte d'Ivoire, similar to the rest of West Africa, and suggest a unique HCV epidemic history in the country.

Full-length genome characterization and genetic relatedness analysis of hepatitis A virus outbreak strains associated with acute liver failure among children.

Clinical infection by hepatitis A virus (HAV) is generally self-limited but in some cases can progress to liver failure. Here, an HAV outbreak investigation among children with acute liver failure in a highly endemic country is presented. In addition, a sensitive method for HAV whole genome amplification and sequencing suitable for analysis of clinical samples is described. In this setting, two fatal cases attributed to acute liver failure and two asymptomatic cases living in the same household were identified. In a second household, one HAV case was observed with jaundice which resolved spontaneously. Partial molecular characterization showed that both households were infected by HAV subtype IA; however, the infecting strains in the two households were different. The HAV outbreak strains recovered from all cases grouped together within cluster IA1, which contains closely related HAV strains from the United States commonly associated with international travelers. Full-genome HAV sequences obtained from the household with the acute liver failure cases were related (genetic distances ranging from 0.01% to 0.04%), indicating a common-source infection. Interestingly, the strain recovered from the asymptomatic household contact was nearly identical to the strain causing acute liver failure. The whole genome sequence from the case in the second household was distinctly different from the strains associated with acute liver failure. Thus, infection with almost identical HAV strains resulted in drastically different clinical outcomes.

Detection of hepatitis C virus transmission by use of DNA mass spectrometry.

The molecular detection of transmission of rapidly mutating pathogens such as hepatitis C virus (HCV) is commonly achieved by assessing the genetic relatedness of strains among infected patients. We describe the development of a novel mass spectrometry (MS)-based approach to identify HCV transmission. MS was used to detect products of base-specific cleavage of RNA molecules obtained from HCV polymerase chain reaction fragments. The MS-peak profiles were found to reflect variation in the HCV genomic sequence and the intrahost composition of the HCV population. Serum specimens originating from 60 case patients from 14 epidemiologically confirmed outbreaks and 25 unrelated controls were tested. Neighbor-joining trees constructed using MS-peak profile-based Hamming distances showed 100% accuracy, and linkage networks constructed using a threshold established from the Hamming distances between epidemiologically unrelated cases showed 100% sensitivity and 99.93% specificity in transmission detection. This MS-based approach is rapid, robust, reproducible, cost-effective, and applicable to investigating transmissions of other pathogens.

Health care-associated hepatitis C virus infections attributed to narcotic diversion.

BACKGROUND: Three cases of genetically related hepatitis C virus (HCV) infection that were unattributable to infection control breaches were identified at a health care facility. OBJECTIVE: To investigate HCV transmission from an HCV-infected health care worker to patients through drug diversion. DESIGN: Cluster and look-back investigations. SETTING: Acute care hospital and affiliated multispecialty clinic. PATIENTS: Inpatients and outpatients during the period of HCV transmission. MEASUREMENTS: Employee work and narcotic dispensing records, blood testing for HCV antibody and RNA, and sequencing of the NS5B gene and the hypervariable region 1 of the E2 gene. RESULTS: 21 employees were recorded as being at work or as retrieving a narcotic from an automated dispensing cabinet in an area where a narcotic was administered to each of the 3 case patients; all employees provided blood samples for HCV testing. One employee was infected with HCV that had more than 95% NS5B sequence homology with the HCV strains of the 3 case patients. Quasi-species analysis showed close genetic relatedness with variants from each of the case patients and more than 97.9% nucleotide identity. The employee acknowledged parenteral opiate diversion. An investigation identified 6132 patients at risk for exposure to HCV because of the drug diversion. Of the 3929 living patients, 3444 (87.7%) were screened for infection. Two additional cases of genetically related HCV infection attributable to the employee were identified. LIMITATION: Of the living patients at risk for HCV exposure, 12.3% were not tested. CONCLUSION: Five cases of HCV infection occurring over 3 to 4 years were attributed to drug diversion by an HCV-infected health care worker. Studies of drug diversion and assessments of strategies to prevent narcotics tampering in all health care settings are needed. PRIMARY FUNDING SOURCE: None.

Epidemic history of hepatitis C virus infection in two remote communities in Nigeria, West Africa.

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.

Temporal variations in the hepatitis C virus intrahost population during chronic infection.

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.

Coordinated evolution among hepatitis C virus genomic sites is coupled to host factors and resistance to interferon.

Machine-learning methods in the form of Bayesian networks (BN), linear projection (LP) and self-organizing tree (SOT) models were used to explore association among polymorphic sites within the HVR1 and NS5a regions of the HCV genome, host demographic factors (ethnicity, gender and age) and response to the combined interferon (IFN) and ribavirin (RBV) therapy. The BN models predicted therapy outcomes, gender and ethnicity with accuracy of 90%, 90% and 88.9%, respectively. The LP and SOT models strongly confirmed associations of the HVR1 and NS5A structures with response to therapy and demographic host factors identified by BN. The data indicate host specificity of HCV evolution and suggest the application of these models to predict outcomes of IFN/RBV therapy.

Hepatitis C virus infections from unsafe injection practices at an endoscopy clinic in Las Vegas, Nevada, 2007-2008.

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.

Artificial neural network for prediction of antigenic activity for a major conformational epitope in the hepatitis C virus NS3 protein.

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_widget.html

Next-Generation Sequencing Reveals Frequent Opportunities for Exposure to Hepatitis C Virus in Ghana.

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.

Genetic host specificity of hepatitis E virus.

Hepatitis E virus (HEV) causes epidemic and sporadic cases of hepatitis worldwide. HEV genotypes 3 (HEV3) and 4 (HEV4) infect humans and animals, with swine being the primary reservoir. The relevance of HEV genetic diversity to host adaptation is poorly understood. We employed a Bayesian network (BN) analysis of HEV3 and HEV4 to detect epistatic connectivity among protein sites and its association with the host specificity in each genotype. The data imply coevolution among ∼70% of polymorphic sites from all HEV proteins and association of numerous coevolving sites with adaptation to swine or humans. BN models for individual proteins and domains of the nonstructural polyprotein detected the host origin of HEV strains with accuracy of 74-93% and 63-87%, respectively. These findings, taken together with lack of phylogenetic association to host, suggest that the HEV host specificity is a heritable and convergent phenotypic trait achievable through variety of genetic pathways (abundance), and explain a broad host range for HEV3 and HEV4.

Hepatitis A virus: host interactions, molecular epidemiology and evolution.

Infection with hepatitis A virus (HAV) is the commonest viral cause of liver disease and presents an important public health problem worldwide. Several unique HAV properties and molecular mechanisms of its interaction with host were recently discovered and should aid in clarifying the pathogenesis of hepatitis A. Genetic characterization of HAV strains have resulted in the identification of different genotypes and subtypes, which exhibit a characteristic worldwide distribution. Shifts in HAV endemicity occurring in different parts of the world, introduction of genetically diverse strains from geographically distant regions, genotype displacement observed in some countries and population expansion detected in the last decades of the 20th century using phylogenetic analysis are important factors contributing to the complex dynamics of HAV infections worldwide. Strong selection pressures, some of which, like usage of deoptimized codons, are unique to HAV, limit genetic variability of the virus. Analysis of subgenomic regions has been proven useful for outbreak investigations. However, sharing short sequences among epidemiologically unrelated strains indicates that specific identification of HAV strains for molecular surveillance can be achieved only using whole-genome sequences. Here, we present up-to-date information on the HAV molecular epidemiology and evolution, and highlight the most relevant features of the HAV-host interactions.

Genetic history of hepatitis C virus in Pakistan.

Hepatitis C virus (HCV) genotype 3a accounts for ∼80% of HCV infections in Pakistan, where ∼10 million people are HCV-infected. Here, we report analysis of the genetic heterogeneity of HCV NS3 and NS5b subgenomic regions from genotype 3a variants obtained from Pakistan. Phylogenetic analyses showed that Pakistani genotype 3a variants were as genetically diverse as global variants, with extensive intermixing. Bayesian estimates showed that the most recent ancestor for genotype 3a in Pakistan was last extant in ∼1896-1914 C.E. (range: 1851-1932). This genotype experienced a population expansion starting from ∼1905 to ∼1970 after which the effective population leveled. Death/birth models suggest that HCV 3a has reached saturating diversity with decreasing turnover rate and positive extinction. Taken together, these observations are consistent with a long and complex history of HCV 3a infection in Pakistan.