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1.  Inferring Viral Dynamics in Chronically HCV Infected Patients from the Spatial Distribution of Infected Hepatocytes 
PLoS Computational Biology  2014;10(11):e1003934.
Chronic liver infection by hepatitis C virus (HCV) is a major public health concern. Despite partly successful treatment options, several aspects of intrahepatic HCV infection dynamics are still poorly understood, including the preferred mode of viral propagation, as well as the proportion of infected hepatocytes. Answers to these questions have important implications for the development of therapeutic interventions. In this study, we present methods to analyze the spatial distribution of infected hepatocytes obtained by single cell laser capture microdissection from liver biopsy samples of patients chronically infected with HCV. By characterizing the internal structure of clusters of infected cells, we are able to evaluate hypotheses about intrahepatic infection dynamics. We found that individual clusters on biopsy samples range in size from infected cells. In addition, the HCV RNA content in a cluster declines from the cell that presumably founded the cluster to cells at the maximal cluster extension. These observations support the idea that HCV infection in the liver is seeded randomly (e.g. from the blood) and then spreads locally. Assuming that the amount of intracellular HCV RNA is a proxy for how long a cell has been infected, we estimate based on models of intracellular HCV RNA replication and accumulation that cells in clusters have been infected on average for less than a week. Further, we do not find a relationship between the cluster size and the estimated cluster expansion time. Our method represents a novel approach to make inferences about infection dynamics in solid tissues from static spatial data.
Author Summary
Around 170 million people worldwide are chronically infected with the hepatitis C virus (HCV). Although partly successful treatment options are available, several aspects of HCV infection dynamics within the liver are still poorly understood. How many hepatocytes are infected during chronic HCV infection? How does the virus propagate, and how do innate immune responses interfere with the spread of the virus? We developed mathematical and computational methods to study liver biopsy samples of patients chronically infected with HCV that were analyzed by single cell laser capture microdissection, to infer the spatial distribution of infected cells. With these methods, we find that infected cells on biopsy sections tend to occur in clusters comprising 4–50 hepatocytes, and, based on their amount of intracellular viral RNA, that these cells have been infected for less than a week. The observed HCV RNA profile within clusters of infected cells suggests that factors such as local immune responses could have shaped cluster expansion and intracellular viral replication. Our methods can be applied to various types of infections in order to infer infection dynamics from spatial data.
doi:10.1371/journal.pcbi.1003934
PMCID: PMC4230741  PMID: 25393308
2.  Constraints on Viral Evolution during Chronic Hepatitis C Virus Infection Arising from a Common-Source Exposure 
Journal of Virology  2012;86(23):12582-12590.
Extraordinary viral sequence diversity and rapid viral genetic evolution are hallmarks of hepatitis C virus (HCV) infection. Viral sequence evolution has previously been shown to mediate escape from cytotoxic T-lymphocyte (CTL) and neutralizing antibody responses in acute HCV infection. HCV evolution continues during chronic infection, but the pressures driving these changes are poorly defined. We analyzed plasma virus sequence evolution in 5.2-kb hemigenomes from multiple longitudinal time points isolated from individuals in the Irish anti-D cohort, who were infected with HCV from a common source in 1977 to 1978. We found phylogenetically distinct quasispecies populations at different plasma time points isolated late in chronic infection, suggesting ongoing viral evolution and quasispecies replacement over time. We saw evidence of early pressure driving net evolution away from a computationally reconstructed common ancestor, known as Bole1b, in predicted CTL epitopes and E1E2, with balanced evolution toward and away from the Bole1b amino acid sequence in the remainder of the genome. Late in chronic infection, the rate of evolution toward the Bole1b sequence increased, resulting in net neutral evolution relative to Bole1b across the entire 5.2-kb hemigenome. Surprisingly, even late in chronic infection, net amino acid evolution away from the infecting inoculum sequence still could be observed. These data suggest that, late in chronic infection, ongoing HCV evolution is not random genetic drift but rather the product of strong pressure toward a common ancestor and concurrent net ongoing evolution away from the inoculum virus sequence, likely balancing replicative fitness and ongoing immune escape.
doi:10.1128/JVI.01440-12
PMCID: PMC3497661  PMID: 22973048
3.  Computational Reconstruction of Bole1a, a Representative Synthetic Hepatitis C Virus Subtype 1a Genome 
Journal of Virology  2012;86(10):5915-5921.
Hepatitis C virus (HCV) research is hampered by the use of arbitrary representative isolates in cell culture and immunology. The most replicative isolate in vitro is a subtype 2a virus (JFH-1); however, genotype 1 is more prevalent worldwide and represents about 70% of infections in the United States, and genotypes differ from one another by 31% to 33% at the nucleotide level. For phylogenetic and immunologic analyses, viruses H77 and HCV-1 (both subtype 1a) are commonly used based on their historic importance. In an effort to rationally design a representative subtype 1a virus (Bole1a), we used Bayesian phylogenetics, ancestral sequence reconstruction, and covariance analysis on a curated set of 390 full-length human HCV 1a sequences from GenBank. By design, Bole1a contains variations present in widely circulating strains and matches more epitope-sized peptides in a full-genome comparison to subtype 1a isolates than any other sequence studied. Parallel analyses confirm that selected epitopes from the Bole1a genome were able to elicit a robust T cell response. In a proof of concept for infectivity, the envelope genes (E1 and E2) of Bole1a were expressed in an HIV pseudoparticle system containing HCV envelope genes and HIV nonenvelope genes with luciferase expression. The resulting Bole1a pseudoparticle robustly infected Hep3B cells. In this study, we demonstrate that a rationally designed, fully synthetic HCV genome contains representative epitopes and envelope genes that assemble properly and mediate entry into target cells.
doi:10.1128/JVI.05959-11
PMCID: PMC3347276  PMID: 22438535
4.  Use of Laser Capture Microdissection to Map Hepatitis C Virus–Positive Hepatocytes in Human Liver 
Gastroenterology  2013;145(6):1404-13.e1-10.
BACKGROUND & AIMS
Hepatitis C virus (HCV) predominantly infects hepatocytes, but many hepatocytes are not infected; studies have shown that HCV antigens cluster within the liver. We investigated spatial distribution and determinants of HCV replication in human liver samples.
METHODS
We analyzed liver samples from 4 patients with chronic HCV infection (genotype 1, Metavir scores 0–1) to estimate the proportion of infected hepatocytes and the amount of HCV viral RNA (vRNA) per cell. Single-cell laser capture microdissection was used to capture more than 1000 hepatocytes in grids, to preserve geometric relationships. HCV vRNA and interferon-induced transmembrane protein 3 (IFITM3) messenger RNA (the transcript of an interferon-stimulated gene) were measured in the same hepatocytes by quantitative polymerase chain reaction and assembled in maps to identify areas of high and low HCV replication.
RESULTS
Patients’ serum levels of HCV RNA ranged from 6.87 to 7.40 log10 IU/mL; the proportion of HCV-infected hepatocytes per person ranged from 21% to 45%, and the level of vRNA ranged from 1 to 50 IU/hepatocyte. Infection was not random; we identified clustering of HCV-positive hepatocytes using infected-neighbor analysis (P < .0005) and distance to the kth nearest neighbor compared with random distributions, obtained by bootstrap simulations (P < .02). Hepatocytes that expressed IFITM3 did not appear to cluster and were largely HCV negative.
CONCLUSIONS
We used single-cell laser capture and high-resolution analysis to show that in human liver HCV infects hepatocytes in nonrandom clusters, whereas expression of antiviral molecules is scattered among hepatocytes. These findings show that quantitative single-cell RNA measurements can be used to estimate the abundance of HCV vRNA per infected human hepatocyte and are consistent with cell–cell propagation of infection in the absence of clustered IFITM3.
doi:10.1053/j.gastro.2013.08.034
PMCID: PMC4005338  PMID: 23973767
ISG; Intrahepatic Infection; Virology; scLCM
5.  The Role of Viral Introductions in Sustaining Community-Based HIV Epidemics in Rural Uganda: Evidence from Spatial Clustering, Phylogenetics, and Egocentric Transmission Models 
PLoS Medicine  2014;11(3):e1001610.
Using different approaches to investigate HIV transmission patterns, Justin Lessler and colleagues find that extra-community HIV introductions are frequent and likely play a role in sustaining the epidemic in the Rakai community.
Please see later in the article for the Editors' Summary
Background
It is often assumed that local sexual networks play a dominant role in HIV spread in sub-Saharan Africa. The aim of this study was to determine the extent to which continued HIV transmission in rural communities—home to two-thirds of the African population—is driven by intra-community sexual networks versus viral introductions from outside of communities.
Methods and Findings
We analyzed the spatial dynamics of HIV transmission in rural Rakai District, Uganda, using data from a cohort of 14,594 individuals within 46 communities. We applied spatial clustering statistics, viral phylogenetics, and probabilistic transmission models to quantify the relative contribution of viral introductions into communities versus community- and household-based transmission to HIV incidence. Individuals living in households with HIV-incident (n = 189) or HIV-prevalent (n = 1,597) persons were 3.2 (95% CI: 2.7–3.7) times more likely to be HIV infected themselves compared to the population in general, but spatial clustering outside of households was relatively weak and was confined to distances <500 m. Phylogenetic analyses of gag and env genes suggest that chains of transmission frequently cross community boundaries. A total of 95 phylogenetic clusters were identified, of which 44% (42/95) were two individuals sharing a household. Among the remaining clusters, 72% (38/53) crossed community boundaries. Using the locations of self-reported sexual partners, we estimate that 39% (95% CI: 34%–42%) of new viral transmissions occur within stable household partnerships, and that among those infected by extra-household sexual partners, 62% (95% CI: 55%–70%) are infected by sexual partners from outside their community. These results rely on the representativeness of the sample and the quality of self-reported partnership data and may not reflect HIV transmission patterns outside of Rakai.
Conclusions
Our findings suggest that HIV introductions into communities are common and account for a significant proportion of new HIV infections acquired outside of households in rural Uganda, though the extent to which this is true elsewhere in Africa remains unknown. Our results also suggest that HIV prevention efforts should be implemented at spatial scales broader than the community and should target key populations likely responsible for introductions into communities.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
About 35 million people (25 million of whom live in sub-Saharan Africa) are currently infected with HIV, the virus that causes AIDS, and about 2.3 million people become newly infected every year. HIV destroys immune system cells, leaving infected individuals susceptible to other infections. HIV infection can be controlled by taking antiretroviral drugs (antiretroviral therapy, or ART) daily throughout life. Although originally available only to people living in wealthy countries, recent political efforts mean that 9.7 million people in low- and middle-income countries now have access to ART. However, ART does not cure HIV infection, so prevention of viral transmission remains extremely important. Because HIV is usually transmitted through unprotected sex with an infected partner, individuals can reduce their risk of infection by abstaining from sex, by having one or a few partners, and by using condoms. Male circumcision also reduces HIV transmission. In addition to reducing illness and death among HIV-positive people, ART also reduces HIV transmission.
Why Was This Study Done?
Effective HIV control requires an understanding of how HIV spreads through sexual networks. These networks include sexual partnerships between individuals in households, between community members in different households, and between individuals from different communities. Local sexual networks (household and intra-community sexual partnerships) are sometimes assumed to be the dominant driving force in HIV spread in sub-Saharan Africa, but are viral introductions from sexual partnerships with individuals outside the community also important? This question needs answering because the effectiveness of interventions such as ART as prevention partly depends on how many new infections in an intervention area are attributable to infection from partners residing in that area and how many are attributable to infection from partners living elsewhere. Here, the researchers use three analytical methods—spatial clustering statistics, viral phylogenetics, and egocentric transmission modeling—to ask whether HIV transmission in rural Uganda is driven predominantly by intra-community sexual networks. Spatial clustering analysis uses the geographical coordinates of households to measure the tendency of HIV-infected people to cluster spatially at scales consistent with community transmission. Viral phylogenetic analysis examines the genetic relatedness of viruses; if transmission is through local networks, viruses in newly infected individuals should more closely resemble viruses in other community members than those in people outside the community. Egocentric transmission modelling uses information on the locations of recent sexual partners to estimate the proportions of new transmissions from household, intra-community, and extra-community partners.
What Did the Researchers Do and Find?
The researchers applied their three analytical methods to data collected from 14,594 individuals living in 46 communities (governmental administrative units) in Rakai District, Uganda. Spatial clustering analysis indicated that individuals who lived in households with individuals with incident HIV (newly diagnosed) or prevalent HIV (previously diagnosed) were 3.2 times more likely than the general population to be HIV-positive themselves. Spatial clustering outside households was relatively weak, however, and was confined to distances of less than half a kilometer. Viral phylogenetic analysis indicated that 44% of phylogenetic clusters (viruses with related genetic sequences found in more than one individual) were within households, but that 40% of clusters crossed community borders. Finally, analysis of the locations of self-reported sexual partners indicated that 39% of new viral transmissions occurred within stable household partnerships, but that among people newly infected by extra-household partners, nearly two-thirds were infected by partners from outside their community.
What Do These Findings Mean?
The results of all three analyses suggest that HIV introductions into communities are frequent and are likely to play an important role in sustaining HIV transmission in the Rakai District. Specifically, within this rural HIV-endemic region (a region where HIV infection is always present), viral introductions combined with intra-household transmission account for the majority of new infections, although community-based sexual networks also play a critical role in HIV transmission. These findings may not be generalizable to the broader Ugandan population or to other regions of Africa, and their accuracy is likely to be limited by the use of self-reported sexual partner data. Nevertheless, these findings indicate that the dynamics of HIV transmission in rural Uganda (and probably elsewhere) are complex. Consequently, to halt the spread of HIV, prevention efforts will need to be implemented at spatial scales broader than individual communities, and key populations that are likely to introduce HIV into communities will need to be targeted.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001610.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS, and summaries of recent research findings on HIV care and treatment
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on HIV and AIDS in Uganda and on HIV prevention strategies (in English and Spanish)
The UNAIDS Report on the Global AIDS Epidemic 2013 provides up-to-date information about the AIDS epidemic and efforts to halt it
The Center for AIDS Prevention Studies (University of California, San Francisco) has a fact sheet about sexual networks and HIV prevention
Wikipedia provides information on spatial clustering analysis (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
A PLOS Computational Biology Topic Page (a review article that is a published copy of record of a dynamic version of the article as found in Wikipedia) about viral phylodynamics is available
Personal stories about living with HIV/AIDS are available through Avert, NAM/aidsmap, and Healthtalkonline
doi:10.1371/journal.pmed.1001610
PMCID: PMC3942316  PMID: 24595023
6.  Genetic divergence of HCV: The role of HIV-related immunosuppression 
Background
We tested the hypothesis that HIV-related immunosuppression alters the host-hepatitis C virus (HCV) interaction, resulting in fewer amino acid changing substitutions in HCV viral variants. Higher HCV RNA levels in persons coinfected with HIV compared with HCV infection alone suggests increased viral replication. If this increase is dependent on decreased immune selective pressure, then a reduced rate of nucleotide changes resulting in amino acid replacements (nonsynonymous changes, dN), would be expected.
Methods
We investigated HCV envelope sequences over time in 79 persons with chronic HCV infection that were HIV negative (group 1), or HIV positive without (group 2) or with (group 3) severe immunodeficiency. We amplified a 1026 nucleotide region of the HCV genome, which encodes a portion of the envelope glycoproteins E1 and E2, including HVR-1 for direct sequence analysis.
Results
The overall divergence between paired sequences, dS, dN, and dN/dS all showed no significant differences among the three groups.
Conclusions
By measuring nucleotide substitutions in HCV sequences over time, we found no significant differences in the genetic divergence between HCV monoinfected control subjects and HIV/HCV coinfected subjects with various levels of immunodeficiency as measured by CD4+ T cell counts.
doi:10.1097/QAI.0b013e3181869a6f
PMCID: PMC3071283  PMID: 18769357
7.  Selection Pressure from Neutralizing Antibodies Drives Sequence Evolution during Acute Infection with Hepatitis C Virus 
Gastroenterology  2009;136(7):2377-2386.
Background & Aims
Despite recent characterization of hepatitis C virus-specific neutralizing antibodies, it is not clear to what extent immune pressure from neutralizing antibodies drives viral sequence evolution in vivo. This lack of understanding is particularly evident in acute infection, the phase when elimination or persistence of viral replication is determined and during which the importance of the humoral immune response has been largely discounted.
Methods
We analyzed envelope glycoprotein sequence evolution, and neutralization of sequential autologous hepatitis C virus pseudoparticles in eight individuals throughout acute infection.
Results
Amino acid substitutions occurred throughout the envelope genes, primarily within the hypervariable region 1 of E2. When individualized pseudoparticles expressing sequential envelope sequences were used to measure neutralization by autologous sera, antibodies neutralizing earlier sequence variants were detected at earlier time points than antibodies neutralizing later variants, indicating clearance and evolution of viral variants in response to pressure from neutralizing antibodies. To demonstrate the effects of amino acid substitution on neutralization, site-directed mutagenesis of a pseudoparticle envelope sequence revealed amino acid substitutions in hypervariable region 1 that were responsible for a dramatic decrease in neutralization sensitivity over time. In addition, high-titer neutralizing antibodies peaked at the time of viral clearance in all spontaneous resolvers, while chronically evolving subjects displayed low-titer or absent neutralizing antibodies throughout early acute infection.
Conclusions
These findings indicate that during acute hepatitis C virus infection in vivo, virus-specific neutralizing antibodies drive sequence evolution and, in some individuals, play a role in determining the outcome of infection.
doi:10.1053/j.gastro.2009.02.080
PMCID: PMC2895772  PMID: 19303013
8.  Maternal Neutralizing Antibody and Transmission of HCV to her Infant 
The Journal of infectious diseases  2008;198(11):1651-1655.
To determine if lower levels of hepatitis C virus (HCV)-specific neutralizing antibodies (nAb) are associated with an increased risk of mother-to-child transmission (MTCT) of HCV, anti-HCV nAb titers were assessed in 63 mothers co-infected with HCV and human immunodeficiency virus type 1 (HIV). Among the mothers, 16 transmitted HCV to their infant but no difference was detected between the ability of maternal plasma from transmitters and non-transmitters to neutralize heterologous HCV pseudoparticles (median nAb titer 1:125 vs. 1:100, P=0.23). In the setting of HIV/HCV co-infection, we found no evidence that anti-HCV nAbs are associated with prevention of MTCT of HCV.
doi:10.1086/593067
PMCID: PMC2777710  PMID: 18928374
HCV; HIV; mother-to-infant transmission; perinatal transmission; hepatitis C virus; neutralizing antibody; HCVpp; MTCT
9.  Comprehensive Analyses of CD8+ T Cell Responses during Longitudinal Study of Acute Human Hepatitis C 
Hepatology (Baltimore, Md.)  2005;42(1):104-112.
We comprehensively studied the cellular immune response during acute human hepatitis C virus (HCV) infection by monthly prospective sampling of persons with high risk of infection. In 19 of 23 subjects, interferon-gamma secreting T cells specific for one or more peptides spanning the entire HCV polyprotein were detected 1–3 months after infection. The median time to development of interferon gamma responses to HCV peptides was 33 days (range 29 to 50 days), and these responses peaked between 180 and 360 days. Nineteen subjects had sufficient follow-up to determine outcome, with 15 (79%) developing persistent viremia and 4 (21%) clearing viremia spontaneously. Of those with progression to chronic infection and detectable T cell responses, all lost recognition of one or more antigens recognized during acute infection and the median reduction in the magnitude of responses was 85%. Most significantly, despite ongoing viremia those who had persistent infection did not develop new epitope specificities after the first six months of infection. In conclusion, these results suggest that in the majority of individuals, the CD8+ T cell responses generated early in HCV infection decline in peripheral blood and are not replaced with new responses.
doi:10.1002/hep.20749
PMCID: PMC2759395  PMID: 15962289
Viral; Immunology; HCV; CD8; ELISpot
10.  Laser Captured Hepatocytes Show Association of BCHE Loss and Fibrosis Progression in Hepatitis C Infected Drug Users 
Hepatology (Baltimore, Md.)  2012;56(2):544-554.
Chronic hepatitis C virus (HCV) infection is complicated by hepatic fibrosis. Hypothesizing that early fibrogenic signals may originate in cells susceptible to HCV infection, hepatocyte gene expression was analyzed from persons with chronic HCV at different stages of liver fibrosis. Four HCV-infected subjects with pre-cirrhotic liver fibrosis (Ishak fibrosis 3–5) were matched for age, race, and gender to five HCV-infected subjects with no evidence of fibrosis (Ishak fibrosis 0). Hepatocytes from each subject were isolated from liver biopsies using laser capture microdissection. Transcriptome profiling was performed on hepatocyte RNA using hybridization arrays. We found that hepatocytes in pre-cirrhotic fibrosis were depleted for genes involved in small molecule and drug metabolism, especially butyrylcholinesterase (BCHE), a gene involved in the metabolism of drugs of abuse. Differential expression of BCHE was validated in the same tissues and cross-sectionally in an expanded cohort of 143 HCV-infected individuals. In a longitudinal study, serum BCHE activity were already decreased at study inception in 19 fibrosis progressors compared to 20 fibrosis non-progressors (p<0.05). Non-progressors also had decreased BCHE activity over time compared to initial values, but these evolved a median (range) 8.6 (7.8–11.4) years after the study period inception(p<0.05). Laser captured portal tracts were enriched for immune related genes when compared to hepatocytes but pre-cirrhotic livers lost this enrichment.
Conclusion
Overall, we found that chronic HCV is associated with hepatocyte BCHE loss years before hepatic synthetic function is impaired. These results indicate that BCHE may be involved in the pathogenesis of HCV-related fibrosis among injection drug users.
doi:10.1002/hep.25655
PMCID: PMC3388175  PMID: 22331678
butyrylcholinesterase; Hepacivirus; chronic HCV infection; laser capture microdissection; portal tract
11.  Spontaneous Clearance of Primary Acute Hepatitis C Virus Infection Correlated with High Initial Viral RNA Level and Rapid HVR1 Evolution 
Hepatology (Baltimore, Md.)  2012;55(6):1684-1691.
Objective/Aims
To determine whether early viral dynamics and evolution predict outcome of primary acute hepatitis C virus (HCV) infection.
Methods
HCV- and HIV-negative injection drug users were enrolled prospectively and followed monthly to identify acute HCV infection using RNA detection. Subjects with more than one month between HCV RNA negative and positive visits were excluded to ensure stringent acute infection. Differences in medians of log-transformed viral RNA levels and evolutionary rates in each gene of a 5’-hemigenomic amplicon were assessed using the Mann-Whitney Rank Sum test. Correlation coefficient was calculated using Spearman Rank Order.
Results
Initial viremia level was 50-fold higher in subjects with spontaneous clearance (compared with persistence) of primary acute HCV infection (median 7.1 versus 5.4 log10 IU/mL, P=0.002). Initial viremia level in subjects with IL-28B-C allele and clearance was higher than that in subjects with IL-28B-T allele and persistence (P=0.001). Evolutionary rates in the hypervariable region 1 (HVR1) region of E2 gene were significantly higher in self-resolvers than those in persistence subjects during early infection, whereas other genes/regions had comparable rates. All major substitutions in HVR1 in persistence subjects were convergent changes whereas over the same time interval clearance subjects displayed divergent evolution, indicating different immune responses between the two groups.
Conclusions
Spontaneous clearance of acute HCV infection is predicted by high initial viremia as well as favorable IL-28B genotype, and associated with rapid envelope sequence evolution. This linkage of host genetics, viral dynamics, and evolution provides new directions for mechanistic studies.
doi:10.1002/hep.25575
PMCID: PMC3330174  PMID: 22234804
12.  Immunogenicity and cross-reactivity of a representative ancestral sequence in HCV infection1 
Vaccines designed to prevent or to treat hepatitis C viral infection must achieve maximum cross reactivity against widely divergent circulating strains. Rational approaches for sequence selection to maximize immunogenicity and minimize genetic distance across circulating strains may enhance vaccine induction of optimal cytotoxic T cell responses. We assessed T cell recognition of potential hepatitis C virus vaccine sequences generated using three rational approaches: 1) combining epitopes with predicted tight binding to the major histocompatibility complex (MHC), 2) consensus sequence (most common amino acid at each position), and 3) representative ancestral sequence that had been derived using Bayesian phylogenetic tools. No correlation was seen between peptide MHC binding affinity and frequency of recognition as measured by an interferon-gamma T cell response in human leukocyte antigen-matched HCV infected individuals. Peptides encoding representative, consensus, and natural variant sequences were then tested for the capacity to expand CD8 T cell populations and to elicit cross-reactive CD8 T cell responses. CD8+ T cells expanded with representative sequence HCV generally more broadly and robustly recognized highly diverse circulating HCV strains than T cell expanded with either consensus sequence or naturally occurring sequence variants. These data support the use of representative sequence in HCV vaccine design.
doi:10.4049/jimmunol.1103008
PMCID: PMC3345099  PMID: 22508927
Animals-human; T cells; Infections-viral; Processes-vaccination
13.  Divergent and convergent evolution after a common-source outbreak of hepatitis C virus 
The Journal of Experimental Medicine  2005;201(11):1753-1759.
The genomic sequences of viruses that are highly mutable and cause chronic infection tend to diverge over time. We report that these changes represent both immune-driven selection and, in the absence of immune pressure, reversion toward an ancestral consensus. Sequence changes in hepatitis C virus (HCV) structural and nonstructural genes were studied in a cohort of women accidentally infected with HCV in a rare common-source outbreak. We compared sequences present in serum obtained 18–22 yr after infection to sequences present in the shared inoculum and found that HCV evolved along a distinct path in each woman. Amino acid substitutions in known epitopes were directed away from consensus in persons having the HLA allele associated with that epitope (immune selection), and toward consensus in those lacking the allele (reversion). These data suggest that vaccines for genetically diverse viruses may be more effective if they represent consensus sequence, rather than a human isolate.
doi:10.1084/jem.20050122
PMCID: PMC2213258  PMID: 15939791
14.  Hepatitis C virus epitope exposure and neutralization by antibodies is affected by time and temperature 
Virology  2011;422(2):174-184.
A recent study with flaviviruses suggested that structural dynamics of the virion impact antibody neutralization via exposure of ostensibly cryptic epitopes. To determine whether this holds true for the distantly related hepatitis C virus (HCV), whose neutralizing epitopes may be obscured by a glycan shield, apolipoprotein interactions, and the hypervariable region on the E2 envelope protein, we assessed how time and temperature of pre-incubation altered monoclonal antibody (MAb) neutralization of HCV. Notably, several MAbs showed increased inhibitory activity when pre-binding was performed at 37°C or after longer pre-incubation periods, and a corresponding loss-of-neutralization was observed when pre-binding was performed at 4°C. A similar profile of changes was observed with acute and chronic phase sera from HCV-infected patients. Our data suggest that time and temperature of incubation modulate epitope exposure on the conformational ensembles of HCV virions and thus, alter the potency of antibody neutralization.
doi:10.1016/j.virol.2011.10.023
PMCID: PMC3356770  PMID: 22078164
antibody; neutralization; hepatitis C; virion; structure
15.  Analysis of Genetic Linkage of HIV From Couples Enrolled in the HIV Prevention Trials Network 052 Trial 
The Journal of Infectious Diseases  2011;204(12):1918-1926.
Background. The HIV Prevention Trials Network (HPTN) 052 trial demonstrated that early initiation of antiretroviral therapy (ART) reduces human immunodeficiency virus (HIV) transmission from HIV-infected adults (index participants) to their HIV-uninfected sexual partners. We analyzed HIV from 38 index-partner pairs and 80 unrelated index participants (controls) to assess the linkage of seroconversion events.
Methods. Linkage was assessed using phylogenetic analysis of HIV pol sequences and Bayesian analysis of genetic distances between pol sequences from index-partner pairs and controls. Selected samples were also analyzed using next-generation sequencing (env region).
Results. In 29 of the 38 (76.3%) cases analyzed, the index was the likely source of the partner’s HIV infection (linked). In 7 cases (18.4%), the partner was most likely infected from a source other than the index participant (unlinked). In 2 cases (5.3%), linkage status could not be definitively established.
Conclusions. Nearly one-fifth of the seroconversion events in HPTN 052 were unlinked. The association of early ART and reduced HIV transmission was stronger when the analysis included only linked events. This underscores the importance of assessing the genetic linkage of HIV seroconversion events in HIV prevention studies involving serodiscordant couples.
doi:10.1093/infdis/jir651
PMCID: PMC3209811  PMID: 21990420
16.  Molecular Epidemiology of HIV Type 1 in Singapore and Identification of Novel CRF01_AE/B Recombinant Forms 
AIDS Research and Human Retroviruses  2011;27(10):1135-1137.
Abstract
To investigate HIV-1 molecular epidemiology in Singapore, we sequenced portions of three regions of the HIV-1 genome (protease HXB2: 2163 to 2620, gp120 HXB2: 6904 to 7628, and gp41 HXB2: 7817 to 8264) from 212 plasma samples collected between February 2008 and August 2009. From these samples, 109 (51.4%) generated interpretable data in all regions. Sixty-one (56.0%) were identified as CRF01_AE, 26 (23.9%) as subtype B and 14 (12.8%) as possible novel recombinant forms. The main novel recombinant pattern, detected in 13 sequences, had subtype B in protease and gp41 and CRF01_AE in gp120. There was intermixing of subtypes within transmission risk groups. However, 85% of subjects infected with the novel recombinant forms self-identified as men who have sex with men or bisexuals compared with only 41% of individuals infected with CRF01_AE and 62% infected with subtype B (p = 0.001).
doi:10.1089/aid.2010.0364
PMCID: PMC3186691  PMID: 21235306
17.  Hepatitis C virus infection of neuroepithelioma cell lines 
Gastroenterology  2010;139(4):1365-1374.
Background & Aims
Hepatitis C virus (HCV) establishes chronic infections in 3% of the world's population. Infection leads to progressive liver disease; hepatocytes are the major site of viral replication in vivo. However, chronic infection is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. We therefore screened a series of neural and brain-derived cell lines for their ability to support HCV entry and replication.
Methods
We used a panel of neural-derived cell lines, HCV pseudoparticles (HCVpp), and an infectious, HCV JFH-1 cell-culture system (HCVcc) to assess viral tropism.
Results
Two independently derived neuroepithelioma cell lines (SK-N-MC and SK-PN-DW) permitted HCVpp entry. In contrast, several neuroblastoma, glioma, and astrocytoma cell lines were refractory to HCVpp infection. HCVcc infected the neuroepithelioma cell lines and established a productive infection. Permissive neuroepithelioma cells expressed CD81, scavenger receptor BI (SR-BI), and the tight junction proteins Claudin-1 (CLDN1) and occludin, whereas non-permissive neural cell lines lacked CLDN1 and in some cases SR-BI. HCVpp infection of the neuroepithelioma cells was neutralized by antibodies to CD81, SR-BI, CLDN1 and HCV E2. Furthermore, anti-CD81, interferon and the anti-NS3 protease inhibitor VX-950 significantly reduced HCVcc infection of neuroepithelioma and hepatoma cells.
Conclusions
Neuroepithelioma-derived cell lines express functional receptors that support HCV entry at comparable levels to that of hepatoma cells. HCV infection in vitro is not restricted to hepatic-derived cells, so HCV might infect cells of the CNS in vivo.
doi:10.1053/j.gastro.2010.06.008
PMCID: PMC3298458  PMID: 20538002
OCLN; neurotropism; brain; therapy; replicon; Huh-7; VX-950
18.  The transhepatic endotoxin gradient is present despite liver cirrhosis and is attenuated after transjugular portosystemic shunt (TIPS). 
BMC Gastroenterology  2011;11:107.
Background
Translocation of gut-derived bacterial products such as endotoxin is a major problem in liver cirrhosis.
Methods
To assess the hepatic clearance of bacterial products in individuals with cirrhosis, we tested concentrations of Gram-negative bacterial lipopolysaccharide (LPS), LPS-binding protein (LBP), and the precursor of nitric oxide (NO), L-arginine, in a cohort of 8 stable patients with liver cirrhosis before and after elective transjugular portosystemic shunt (TIPS) implantation, including central venous, hepatic venous, and portal venous measurements.
Results
Using an adapted LPS assay, we detected high portal venous LPS concentrations (mean 1743 ± 819 pg/mL). High concentrations of LPS were detectable in the central venous blood (931 ± 551 pg/mL), as expected in persons with cirrhosis. The transhepatic LPS gradient was found to be 438 ± 287 pg/mL, and 25 ± 12% of portal LPS was cleared by the cirrhotic liver. After TIPS, central venous LPS concentrations increased in the hepatic and central veins, indicating shunting of LPS with the portal blood through the stent. This paralleled a systemic increase of L-arginine, whereas the NO synthase inhibitor asymmetric dimethylarginine (ADMA) remained unchanged, suggesting that bacterial translocation may contribute to the pathogenesis of circulatory dysfunction post-TIPS.
Conclusions
This study provides quantitative estimates of the role of the liver in the pathophysiology of bacterial translocation. The data indicate that the cirrhotic liver retains the capacity for clearance of bacterial endotoxin from the portal venous blood and that TIPS implantation attenuates this clearance. Thus, increased endotoxin concentrations in the systemic circulation provide a possible link to the increased encephalopathy in TIPS patients.
doi:10.1186/1471-230X-11-107
PMCID: PMC3203072  PMID: 21978390
19.  Analysis of HIV Diversity Using a High-Resolution Melting Assay 
Abstract
HIV viruses are usually genetically homogeneous shortly after infection, and become more heterogeneous over time. We developed a high-resolution melting (HRM) assay to analyze HIV diversity without sequencing. Plasma samples from the HIVNET 012 trial were obtained from nine Ugandan mother–infant pairs. DNA amplified from the HIV gag region was analyzed to determine the number of degrees over which the DNA melted (HRM score). HRM gag DNA was also cloned and sequenced (50 clones/mother; 20 clones/infant). The median HRM score for infants (4.3, range 4.2–5.3) was higher than that for control plasmids (3.4, range 3.2–3.8, p < 0.001) and lower than that for mothers (5.7, range 4.4–7.7, p = 0.005, exact Wilcoxon rank sum test). The intraclass correlation coefficient reflecting assay reproducibility was 94% (95% CI: 89–98%). HRM scores were also compared to sequenced-based measures of HIV diversity; higher HRM scores were associated with higher genetic diversity (p < 0.001), complexity (p = 0.009), and Shannon entropy (p = 0.022), but not with length variation (p = 0.111). The HRM assay provides a novel, rapid method for assessing HIV diversity without sequencing. This assay could be applied to any region of the HIV genome or to other genetic systems that exhibit DNA diversity.
doi:10.1089/aid.2009.0259
PMCID: PMC2920076  PMID: 20666583
21.  Spontaneous Control of Primary Hepatitis C Virus Infection and Immunity against Persistent Reinfection 
Gastroenterology  2009;138(1):315-324.
Background & Aims
We followed persons with ongoing hepatitis C virus (HCV) exposure following control of an initial HCV infection to determine whether primary control conferred protection against future persistent infections.
Methods
Twenty-two active injection drug users (IDU) who had cleared a primary hepatitis C viremia for at least 60 days were monitored monthly. Reinfection was defined as the detection of a new hepatitis C virus infection. Protection was assessed based on the magnitude and duration of viremia following reinfection and generation of T-cell and neutralizing antibody (nAb) responses
Results
Reinfection occurred in 11 IDUs (50%) who previously spontaneously controlled primary HCV infection. Although viral clearance occurs in approximately 25% of patients with primary infections, spontaneous viral clearance was observed in 83% of reinfected patients. The duration and maximum level of viremia during subsequent episodes of reinfection were significantly decreased, compared with those of the primary infection in the same subjects. In contrast to chronic infection, reinfection was associated with a significant increase in the breadth of T-cell responses. During acute infection, nAbs against heterologous viral pseudoparticles were detected in 60% of reinfected subjects; cross-reactive nAbs are rarely detected in patients who progress to chronic infection.
Conclusions
HCV reinfection is associated with a reduction in the magnitude and duration of viremia (compared with the initial infection), broadened cellular immune responses, and the generation of cross-reactive humoral responses. These findings are consistent with the development of adaptive immunity that is not sterilizing but protects against chronic disease.
doi:10.1053/j.gastro.2009.09.017
PMCID: PMC2889495  PMID: 19782080
22.  Control of HIV-1 in Elite Suppressors despite Ongoing Replication and Evolution in Plasma Virus▿  
Journal of Virology  2010;84(14):7018-7028.
A subset of HIV-1-infected patients known as elite controllers or suppressors (ES) control the virus naturally. We have previously demonstrated sequence discordance between proviral and plasma gag clones in ES, much of which can be attributed to selective pressure from the host (J. R. Bailey, T. M. Williams, R. F. Siliciano, and J. N. Blankson, J. Exp. Med. 203:1357-1369, 2006). However, it is not clear whether ongoing viral replication continues in ES once the control of viremia has been established or whether selective pressure impacts this evolution. The cytotoxic T-lymphocyte (CTL) response in ES often targets Gag and frequently is superior to that of HIV-1 progressors, partially due to the HLA class I alleles B*57/5801 and B*27, which are overrepresented in ES. We therefore examined longitudinal plasma and proviral gag sequences from HLA-B*57/5801 and -B*27 ES. Despite the highly conserved nature of gag, we observed clear evidence of evolution in the plasma virus, largely due to synonymous substitutions. In contrast, evolution was rare in proviral clones, suggesting that ongoing replication in ES does not permit the significant reseeding of the latent reservoir. Interestingly, there was little continual evolution in CTL epitopes, and we detected de novo CTL responses to autologous viral mutants. Thus, some ES control viremia despite ongoing replication and evolution.
doi:10.1128/JVI.00548-10
PMCID: PMC2898225  PMID: 20444904
23.  HIV-1 Evolution Following Transmission to an HLA-B*5801 Positive Subject 
The Journal of infectious diseases  2009;200(12):1820-1824.
The HIV-1-specific immune responses of HLA-B*57/5801 patients who spontaneously control viral replication serve as an important model for T cell-based HIV-1 vaccines. Determining the breadth of this response and the extent of virologic escape that occur in primary infection in these patients is therefore critical. Here we document the development of mutations in 3 HLA-B*5801 restricted epitopes in gag, nef and pol in an HLA-B*5801 subject with a viral load of only 1159 copies/ml at day 167 post-infection. Thus, relative control of viral replication can be maintained in spite of the rapid development of multiple escape mutations within CTL epitopes.
doi:10.1086/648377
PMCID: PMC2779566  PMID: 19909081
24.  High Diversity of Hepatitis C Viral Quasispecies is Associated with Early Virological Response in Patients undergoing Antiviral Therapy 
Hepatology (Baltimore, Md.)  2009;50(6):1765-1772.
Differential response patterns to optimal antiviral therapy, peginterferon alpha plus ribavirin, are well documented in patients with chronic hepatitis C virus (HCV) infection. Among many factors that may affect therapeutic efficiency, HCV quasispecies (QS) characteristics have been a major focus of previous studies, yielding conflicting results. To obtain a comprehensive understanding of the role of HCV QS in antiviral therapy, we performed the largest-ever HCV QS analysis in 153 patients infected with HCV genotype 1 strains. A total of 4,314 viral clones spanning hypervarible region 1 were produced from these patients during the first 12 weeks of therapy, followed by detailed genetic analyses. Our data showed an exponential distribution pattern of intra-patient QS diversity in this study population in which most patients (63%) had small QS diversity with genetic distance (d) less than 0.2. The group of patients with genetic distance located in the decay region (d>0.53) had a significantly higher early virologic response (EVR) rate (89.5%), which contributed substantially to the overall association between EVR and increased baseline QS diversity. In addition, EVR was linked to a clustered evolutionary pattern in terms of QS dynamic changes.
Conclusion
EVR is associated with elevated HCV QS diversity and complexity, especially in patients with significantly higher HCV genetic heterogeneity.
doi:10.1002/hep.23290
PMCID: PMC2911951  PMID: 19937690
Hepatitis C virus; Quasispecies; Antiviral therapy
25.  Epidemiology of hepatitis C virus infection & liver disease among injection drug users (IDUs) in Chennai, India 
Background & Objectives:
We characterized HCV antibody prevalence, viral persistence, genotype and liver disease prevalence among IDUs in Chennai, India as the study of the association of HIV with each of these states is important and there are no data available.
Methods:
Between 2005-2006, 1158 IDUs were recruited and followed semi-annually. All were tested for HCV antibodies at baseline; a random sample of 400 antibody positives (200 HIV-positive and 200 HIV-negative) were tested for HCV RNA; 13 of these were sequenced. Assessment of asparate amino transferase (AST)-to-platelet ratio index (APRI) was done on 557 IDUs. Prevalence ratios of each outcome were examined.
Results:
Median age was 35 yr; 99 per cent were male. HCV antibody prevalence was 55 per cent and was associated with older age, being unmarried, longer injection history, tattoo and injecting at a dealer’s place. Of the 400 HCV antibody positive IDUs, 281 (70.3%) had persistent infection which was less common among hepatitis B-infected persons but not associated with HIV. Of the 13 samples sequenced, 11 (85%) were HCV genotype 3a. Fibrosis prevalence according to APRI was: HIV/HCV-uninfected, 4 per cent; HIV mono-infected, 3 per cent; HCV mono-infected, 11 per cent; HIV/HCV co-infected, 12 per cent (P<0.001). In addition to being associated with HCV and HIV/HCV, fibrosis prevalence was higher among those drinking alcohol frequently; daily marijuana use was protective.
Interpretation & Conclusions:
Our findings show that IDUs in Chennai have high HCV prevalence and associated disease burden. The burden will increase as access to antiretroviral therapy improves particularly given the high prevalence of HIV, HCV and alcohol use.
PMCID: PMC3102459  PMID: 21245619
APRI; HCV genotype; hepatitis C virus; HIV; injection drug users; liver disease

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