Showing papers by "Peter Simmonds published in 2013"

Identification of Rodent Homologs of Hepatitis C Virus and Pegiviruses

Abstract: Hepatitis C virus (HCV) and human pegivirus (HPgV or GB virus C) are globally distributed and infect 2 to 5% of the human population. The lack of tractable-animal models for these viruses, in particular for HCV, has hampered the study of in- fection, transmission, virulence, immunity, and pathogenesis. To address this challenge, we searched for homologous viruses in small mammals, including wild rodents. Here we report the discovery of several new hepaciviruses (HCV-like viruses) and pegi- viruses (GB virus-like viruses) that infect wild rodents. Complete genome sequences were acquired for a rodent hepacivirus (RHV) found in Peromyscus maniculatus and a rodent pegivirus (RPgV) found in Neotoma albigula. Unique genomic features and phylogenetic analyses confirmed that these RHV and RPgV variants represent several novel virus species in the Hepacivirus andPegivirusgenera within the family Flaviviridae. The genetic diversity of the rodent hepaciviruses exceeded that observed for hepaciviruses infecting either humans or non-primates, leading to new insights into the origin, evolution, and host range of hepaciviruses. The presence of genes, encoded proteins, and translation elements homologous to those found in human hepaci- viruses and pegiviruses suggests the potential for the development of new animal systems with which to model HCV pathogene- sis, vaccine design, and treatment. IMPORTANCE The genetic and biological characterization of animal homologs of human viruses provides insights into the origins of human infections and enhances our ability to study their pathogenesis and explore preventive and therapeutic interventions. Horses are the only reported host of nonprimate homologs of hepatitis C virus (HCV). Here, we report the discovery of HCV- like viruses in wild rodents. The majority of HCV-like viruses were found in deer mice (Peromyscus maniculatus), a small rodent used in laboratories to study viruses, including hantaviruses. We also identified pegiviruses in rodents that are distinct from the pegiviruses found in primates, bats, and horses. These novel viruses may enable the development of small-animal models for HCV, the most common infectious cause of liver failure and hepatocellular carcinoma after hepatitis B virus, and help to explore the health relevance of the highly prevalent human pegiviruses.

The Origin of Hepatitis C Virus

Abstract: The origin of hepatitis C virus (HCV) can be conceptualised at several levels. Firstly, origins might refer to its dramatic spread throughout the Western world and developing countries throughout the twentieth century. As a blood-borne virus, this epidemic was fuelled by new parenteral transmission routes associated with medical treatments, immunisation, blood transfusion and more recently injecting drug use. At another level, however, origins might refer to the immediate sources of HCV associated with its pandemic spread, now identified as areas in Central and West sub-Saharan Africa and South and South East Asia where genetically diverse variants of HCV appear to have circulated for hundreds of years. Going back a final step to the actual source of HCV infection in these endemic areas, non-human primates have been long suspected as harbouring viruses related to HCV with potential cross-species transmission of variants corresponding to the 7 main genotypes into humans. Although there is tempting analogy between this and the clearly zoonotic origin of HIV-1 from chimpanzees in Central Africa, no published evidence to date has been obtained for infection of HCV-like viruses in either apes or Old World monkey species. Indeed, a radical re-think of both the host range and host-specificity of hepaciviruses is now required following the very recent findings of a non-primate hepacivirus (NPHV) in horses and potentially in dogs. Further research on a much wider range of mammals is needed to better understand the true genetic diversity of HCV-like viruses and their host ranges in the search for the ultimate origin of HCV in humans.

Prospective Observational Study of Breast Cancer Treatment Outcomes for UK Women Aged 18–40 Years at Diagnosis: The POSH Study

Abstract: Background: Breast cancer at a young age is associated with poor prognosis. The Prospective Study of Outcomes in Sporadic and Hereditary Breast Cancer (POSH) was designed to investigate factors affecting prognosis in this patient group. Methods: Between 2000 and 2008, 2956 patients aged 40 years or younger were recruited to a UK multicenter prospective observational cohort study (POSH). Details of tumor pathology, disease stage, treatment received, and outcome were recorded. Overall survival (OS) and distant disease-free interval (DDFI) were assessed using Kaplan-Meier curves. All statistical tests were two-sided. Results: Median age of patients was 36 years. Median tumor diameter was 22mm, and 50% of patients had positive lymph nodes; 59% of tumors were grade 3, 33.7% were estrogen receptor (ER) negative, and 24% were human epidermal growth factor receptor 2 (HER2) positive. Five-year OS was higher for patients with ER-positive than ER-negative tumors (85.0%, 95% confidence interval [CI] = 83.2% to 86.7% vs 75.7%, 95% CI = 72.8% to 78.4%; P Conclusions: These results confirm the increased frequency of ER-negative tumors and early relapse in young patients and also demonstrate the equally poor longer-term outlook of young patients who have ER-positive tumors with HER2-negative or -positive disease.

Genetic Variability and the Classification of Hepatitis E Virus

Abstract: The classification of hepatitis E virus (HEV) variants is currently in transition without agreed definitions for genotypes and subtypes or for deeper taxonomic groupings into species and genera that could incorporate more recently characterized viruses assigned to the Hepeviridae family that infect birds, bats, rodents, and fish. These conflicts arise because of differences in the viruses and genomic regions compared and in the methodology used. We have reexamined published sequences and found that synonymous substitutions were saturated in comparisons between and within virus genotypes. Analysis of complete genome sequences or concatenated ORF1/ORF2 amino acid sequences indicated that HEV variants most closely related to those infecting humans can be consistently divided into six genotypes (types 1 to 4 and two additional genotypes from wild boar). Variants isolated from rabbits, closely related to genotype 3, occupy an intermediate position. No consistent criteria could be defined for the assignment of virus subtypes. Analysis of amino acid sequences from these viruses with the more divergent variants from chickens, bats, and rodents in three conserved subgenomic regions (residues 1 to 452 or 974 to 1534 of ORF1 or residues 105 to 458 of ORF2) provided consistent support for a division into 4 groups, corresponding to HEV variants infecting humans and pigs, those infecting rats and ferrets, those from bats, and those from chickens. This approach may form the basis for a future genetic classification of HEV into four species, with the more divergent HEV-like virus from fish (cutthroat trout virus) representing a second genus.

Re-evaluation of the lifetimes of the major CFCs and CH 3 CCl 3 using atmospheric trends

Abstract: . Since the Montreal Protocol on Substances that Deplete the Ozone Layer and its amendments came into effect, growth rates of the major ozone depleting substances (ODS), particularly CFC-11, -12 and -113 and CH3CCl3, have declined markedly, paving the way for global stratospheric ozone recovery. Emissions have now fallen to relatively low levels, therefore the rate at which this recovery occurs will depend largely on the atmospheric lifetime of these compounds. The first ODS measurements began in the early 1970s along with the first lifetime estimates calculated by considering their atmospheric trends. We now have global mole fraction records spanning multiple decades, prompting this lifetime re-evaluation. Using surface measurements from the Advanced Global Atmospheric Gases Experiment (AGAGE) and the National Oceanic and Atmospheric Administration Global Monitoring Division (NOAA GMD) from 1978 to 2011, we estimated the lifetime of CFC-11, CFC-12, CFC-113 and CH3CCl3 using a multi-species inverse method. A steady-state lifetime of 45 yr for CFC-11, currently recommended in the most recent World Meteorological Organisation (WMO) Scientific Assessments of Ozone Depletion, lies towards the lower uncertainty bound of our estimates, which are 544861 yr (1-sigma uncertainty) when AGAGE data were used and 524561 yr when the NOAA network data were used. Our derived lifetime for CFC-113 is significantly higher than the WMO estimates of 85 yr, being 10999121 (AGAGE) and 10997124 (NOAA). New estimates of the steady-state lifetimes of CFC-12 and CH3CCl3 are consistent with the current WMO recommendations, being 11195132 and 11295136 yr (CFC-12, AGAGE and NOAA respectively) and 5.044.925.20 and 5.044.875.23 yr (CH3CCl3, AGAGE and NOAA respectively).

Identification of a Pegivirus (GB Virus-Like Virus) That Infects Horses

Abstract: The recent identification of nonprimate hepaciviruses in dogs and then in horses prompted us to look for pegiviruses (GB virus-like viruses) in these species. Although none were detected in canines, we found widespread natural infection of horses by a novel pegivirus. Unique genomic features and phylogenetic analyses confirmed that the tentatively named equine pegivirus (EPgV) represents a novel species within the Pegivirus genus. We also determined that EPgV causes persistent viremia whereas its clinical significance is undetermined.

Modelling mutational and selection pressures on dinucleotides in eukaryotic phyla –selection against CpG and UpA in cytoplasmically expressed RNA and in RNA viruses

Abstract: Loss of CpG dinucleotides in genomic DNA through methylation-induced mutation is characteristic of vertebrates and plants. However, these and other eukaryotic phyla show a range of other dinucleotide frequency biases with currently uncharacterized underlying mutational or selection mechanisms. We developed a parameterized Markov process to identify what neighbour context-dependent mutations best accounted for patterns of dinucleotide frequency biases in genomic and cytoplasmically expressed mRNA sequences of different vertebrates, other eukaryotic groups and RNA viruses that infect them. Consistently, 11- to 14-fold greater frequencies of the methylation-associated mutation of C to T upstream of G (depicted as C→T,G) than other transitions best modelled dinucleotide frequencies in mammalian genomic DNA. However, further mutations such as G→T,T (5-fold greater than the default transversion rate) were required to account for the full spectrum of dinucleotide frequencies in mammalian sequence datasets. Consistent with modeling predictions for these two mutations, instability of both CpG and CpT dinucleotides was identified through SNP frequency analysis of human DNA sequences. Different sets of context-dependent mutations were modelled in other eukaryotes with non-methylated genomic DNA. In contrast to genomic DNA, best-fit models of dinucleotide frequencies in transcribed RNA sequences expressed in the cytoplasm from all organisms were dominated by mutations that eliminated UpA dinucleotides, observations consistent with cytoplasmically driven selection for mRNA stability. Surprisingly, mRNA sequences from organisms with methylated genomes showed evidence for additional selection against CpG through further context-dependent mutations (eg. C→A,G). Similar mutation or selection processes were identified among single-stranded mammalian RNA viruses; these potentially account for their previously described but unexplained under-representations of CpG and UpA dinucleotides. Methods we have developed identify mutational processes and selection pressures in organisms that provide new insights into nucleotide compositional constraints and a wealth of biochemical and evolutionarily testable predictions for the future.

Whole Genome Pyrosequencing of Rare Hepatitis C Virus Genotypes Enhances Subtype Classification and Identification of Naturally Occurring Drug Resistance Variants

Abstract: Hepatitis C virus (HCV) is an enveloped, positive-strand RNA virus belonging to the Hepacivirus genus in the Flaviviridae family. Seven confirmed genotypes (1–7) are generally distinguished by phylogenetic methods and pair-wise distance calculations [1, 2]. On the basis of full genome nucleotide sequences, HCV genotypes diverged from each other by a pair-wise distance of >30%. Individual genotypes can be further divided into more closely related subtypes that diverged by a pair-wise distance of 15%–30%. All viral genotypes retain their repertoire of colinear structural and nonstructural genes, thereby facilitating preliminary genotype classification on the basis of partial genome sequences of short fragments (approximately 300–400 nucleotides) in the structural core/E1 region and the nonstructural NS5B region [3]. However, full genome sequences remain indispensible for the detection of genome recombination events. While only recombinant HCV genotype 2 k/1b viruses have been found to actively circulate in the population so far [4–7], recombinants of genotypes 2/5, 2b/1b, 2b/1a, and 2i/6p have been detected in single isolates from humans [8–11]. Furthermore, the frequency of HCV intergenotype and intragenotype recombination may be underestimated because of the lack of robust detection methods [12, 13]. Although genotyping based on core/E1 or NS5B sequences has resulted in the provisional classification of a large number of subtype variants within each genotype, at least 1 but preferably ≥2 full genome sequences are required to confirm subtype designation [2, 12]. Accurate genotype and subtype classification is clinically important because major genotypes differ considerably in their response rates to treatment with pegylated interferon and ribavirin and with directly acting antiviral drugs (DAAs) that are designed mainly against genotype 1 isolates. The limited efficacy of DAAs against other genotypes has been shown for genotype 3 [14], and treatment response rates for drug regimens containing boceprevir (an NS3 protease inhibitor) or BMS-790052 (an NS5A inhibitor) were found to vary even between subtypes 1a and 1b [15, 16]. Different baseline frequencies of drug resistance mutations may account for these differences [17], and such variation might become more significant if interferon-free regimens containing 1 or more DAAs could indeed displace the current standard of care [18]. Until clinically tolerable DAAs with high barriers to resistance against the complete spectrum of HCV genotypes become available [18, 19], accurate subtype determination and, possibly, drug resistance profiling of the individual's viral population may guide the optimal choice of drugs. Full genome deep sequencing, as performed in this study, allows the identification of drug resistance mutations across the genome that may exist as dominant or minor variants in treatment-naive patients, thereby informing the design of therapeutic regimens.

Recombination in the evolution of human rhinovirus genomes.

Abstract: Human rhinoviruses (HRV) are highly prevalent human respiratory pathogens that belong to the genus Enterovirus. Although recombination within the coding region is frequent in other picornavirus groups, most evidence of recombination in HRV has been restricted to the 5' untranslated region. We analysed the occurrence of recombination within published complete genome sequences of members of all three HRV species and additionally compared sequences from HRV strains spanning 14 years. HRV-B and HRV-C showed very little evidence of recombination within the coding region. In contrast, HRV-A sequences appeared to have undergone a large number of recombination events, typically involving whole type groups. This suggests that HRV-A may have been subject to extensive recombination during the period of diversification into types. This study demonstrates the rare and sporadic nature of contemporary recombination of HRV strains and contrasts with evidence of extensive recombination within HRV-A and between members of different species during earlier stages in its evolutionary diversification.

Sequencing of the Hepatitis C Virus: A Systematic Review

Abstract: Since the identification of hepatitis C virus (HCV), viral sequencing has been important in understanding HCV classification, epidemiology, evolution, transmission clustering, treatment response and natural history. The length and diversity of the HCV genome has resulted in analysis of certain regions of the virus, however there has been little standardisation of protocols. This systematic review was undertaken to map the location and frequency of sequencing on the HCV genome in peer reviewed publications, with the aim to produce a database of sequencing primers and amplicons to inform future research. Medline and Scopus databases were searched for English language publications based on keyword/MeSH terms related to sequence analysis (9 terms) or HCV (3 terms), plus “primer” as a general search term. Exclusion criteria included non-HCV research, review articles, duplicate records, and incomplete description of HCV sequencing methods. The PCR primer locations of accepted publications were noted, and purpose of sequencing was determined. A total of 450 studies were accepted from the 2099 identified, with 629 HCV sequencing amplicons identified and mapped on the HCV genome. The most commonly sequenced region was the HVR-1 region, often utilised for studies of natural history, clustering/transmission, evolution and treatment response. Studies related to genotyping/classification or epidemiology of HCV genotype generally targeted the 5′UTR, Core and NS5B regions, while treatment response/resistance was assessed mainly in the NS3–NS5B region with emphasis on the Interferon sensitivity determining region (ISDR) region of NS5A. While the sequencing of HCV is generally constricted to certain regions of the HCV genome there is little consistency in the positioning of sequencing primers, with the exception of a few highly referenced manuscripts. This study demonstrates the heterogeneity of HCV sequencing, providing a comprehensive database of previously published primer sets to be utilised in future sequencing studies.

Influence of genome-scale RNA structure disruption on the replication of murine norovirus—similar replication kinetics in cell culture but attenuation of viral fitness in vivo

Abstract: Mechanisms by which certain RNA viruses, such as hepatitis C virus, establish persistent infections and cause chronic disease are of fundamental importance in viral pathogenesis. Mammalian positive-stranded RNA viruses establishing persistence typically possess genome-scale ordered RNA secondary structure (GORS) in their genomes. Murine norovirus (MNV) persists in immunocompetent mice and provides an experimental model to functionally characterize GORS. Substitution mutants were constructed with coding sequences in NS3/4- and NS6/7-coding regions replaced with sequences with identical coding and (di-)nucleotide composition but disrupted RNA secondary structure (F1, F2, F1/F2 mutants). Mutants replicated with similar kinetics to wild-type (WT) MNV3 in RAW264.7 cells and primary macrophages, exhibited similar (highly restricted) induction and susceptibility to interferon-coupled cellular responses and equal replication fitness by serial passaging of co-cultures. In vivo, both WT and F1/F2 mutant viruses persistently infected mice, although F1, F2 and F1/F2 mutant viruses were rapidly eliminated 1–7 days post-inoculation in competition experiments with WT. F1/F2 mutants recovered from tissues at 9 months showed higher synonymous substitution rates than WT and nucleotide substitutions that potentially restored of RNA secondary structure. GORS plays no role in basic replication of MNV but potentially contributes to viral fitness and persistence in vivo.

Evolution of CD8+ T Cell Responses after Acute PARV4 Infection

Abstract: PARV4 is a small DNA human virus that is strongly associated with hepatitis C virus (HCV) and HIV infections. The immunologic control of acute PARV4 infection has not been previously described. We define the acute onset of PARV4 infection and the characteristics of the acute-phase and memory immune responses to PARV4 in a group of HCV- and HIV-negative, active intravenous drug users. Ninety-eight individuals at risk of blood-borne infections were tested for PARV4 IgG. Gamma interferon enzyme-linked immunosorbent spot assays, intracellular cytokine staining, and a tetrameric HLA-A2-peptide complex were used to define the T cell populations responding to PARV4 peptides in those individuals who acquired infection during the study. Thirty-five individuals were found to be PARV4 seropositive at the end of the study, eight of whose baseline samples were found to be seronegative. Persistent and functional T cell responses were detected in the acute infection phase. These responses had an active, mature, and cytotoxic phenotype and were maintained several years after infection. Thus, PARV4 infection is common in individuals exposed to blood-borne infections, independent of their HCV or HIV status. Since PARV4 elicits strong, broad, and persistent T cell responses, understanding of the processes responsible may prove useful for future vaccine design.

Hepatitis E virus mixed infection in immunocompetent patient.

Abstract: We detected 2 hepatitis E virus (HEV) strains in an acutely infected immunocompetent patient. Two populations of genotype 3 virus were observed in the hypervariable regions and open reading frames 2 and 3, indicating multiple infection with hepatitis E virus. Persons with mixed infections may provide the opportunity for virus recombination.

A short bifunctional element operates to positively or negatively regulate ESAG9 expression in different developmental forms of Trypanosoma brucei

Abstract: In their mammalian host trypanosomes generate ‘stumpy’ forms from proliferative ‘slender’ forms as an adaptation for transmission to their tsetse fly vector This transition is characterised by the repression of many genes while quiescent stumpy forms accumulate during each wave of parasitaemia However, a subset of genes are upregulated either as an adaptation for transmission or to sustain infection chronicity Among this group are ESAG9 proteins, whose genes were originally identified as a component of some telomeric variant surface glycoprotein gene expression sites, although many members of this diverse family are also transcribed elsewhere in the genome ESAG9 genes are among the most highly regulated genes in transmissible stumpy forms, encoding a group of secreted proteins of cryptic function To understand their developmental silencing in slender forms and activation in stumpy forms, the post-transcriptional control signals for a well conserved ESAG9 gene have been mapped This identified a precise RNA sequence element of 34 nucleotides that contributes to gene expression silencing in slender forms but also acts positively, activating gene expression in stumpy forms We predict that this bifunctional RNA sequence element is targeted by competing negative and positive regulatory factors in distinct developmental forms of the parasite Analysis of the 3′UTR regulatory regions flanking the highly diverse ESAG9 family reveals that the linear regulatory sequence is not highly conserved, suggesting that RNA structure is important for interactions with regulatory proteins

Rosavirus: the prototype of a proposed new genus of the Picornaviridae family.

Abstract: We describe a 8,724-nucleotide-long picornavirus genome encoding a single 2,470-aa polyprotein obtained from the feces of a wild mouse. Rosavirus is genetically closest to the double ORF Dicipivirus found in canine feces that is currently the only picornavirus with a second internal ribosome entry site (IRES). Of note, a section of rosavirus' 5'UTR showed strong sequence and structural conservation with the type II IRES from the Parechovirus and Hungarovirus genera possibly reflecting exchange of genetic modules between genera. Based on genetic distance criteria rosavirus qualifies as prototype of a new genus of the Picornaviridae family.

Interannual fluctuations in the seasonal cycle of nitrous oxide and chlorofluorocarbons due to the Brewer-Dobson circulation

Abstract: [1] The tropospheric seasonal cycles of N2O, CFC-11 (CCl3F), and CFC-12 (CCl2F2) are influenced by atmospheric dynamics. The interannually varying summertime minima in mole fractions of these trace gases have been attributed to interannual variations in mixing of stratospheric air (depleted in CFCs and N2O) with tropospheric air with a few months lag. The amount of wave activity that drives the stratospheric circulation and influences the winter stratospheric jet and subsequent mass transport across the tropopause appears to be the primary cause of this interannual variability. We relate the observed seasonal minima of species at three Northern Hemisphere sites (Mace Head, Ireland; Trinidad Head, U.S.; and Barrow, Alaska) with the behavior of the winter stratospheric jet. As a result, a good correlation is obtained between zonal winds in winter at 10 hPa, 58°N–68°N, and the detrended seasonal minima in the stratosphere-influenced tracers. For these three tracers, individual Pearson correlation coefficients (r) between 0.51 and 0.71 were found, with overall correlations of between 0.67 and 0.77 when “composite species” were considered. Finally, we note that the long-term observations of CFCs and N2O in the troposphere provide an independent monitoring method complementary to satellite data. Furthermore, they could provide a useful observational measure of the strength of stratosphere-troposphere exchange and, thus, could be used to monitor any long-term trend in the Brewer-Dobson circulation which is predicted by climate models to increase over the coming decades.

The non-primate hepacivirus 5′ untranslated region possesses internal ribosomal entry site activity

Abstract: The 5′ untranslated region (5′UTR) of the recently described non-primate hepacivirus (NPHV) contains a region with sequence homology to the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) and GB virus B (GBV-B). Here, we demonstrated internal translation initiation by the NPHV 5′UTR in a bicistronic vector. An RNA stem–loop upstream of the NPHV IRES was structurally distinct from corresponding regions in HCV and GBV-B, and was not required for IRES function. Insertion of the NPHV stem–loop into the corresponding region of the HCV 5′UTR within the HCV subgenomic replicon significantly impaired RNA replication, indicating that long-range interactions between the 5′UTR and cis-acting downstream elements within the NPHV genome are not interchangeable with those of HCV. Despite similarities in IRES structure and function between hepaciviruses, replication elements in the NPHV 5′UTR appear functionally distinct from those of HCV.

Frequency and pathogenicity of rhinovirus associated pulmonary exacerbations in patients with cystic fibrosis

Abstract: Objectives: Human rhinovirus (HRV) is associated with pulmonary exacerbations in patients with CF. With sensitive RT-PCR techniques HRV is readily detected and new strains are being identified. In asthma, children with HRV group C have been shown to have more severe exacerbations. The aim of this project was to establish the frequency of each HRV group during exacerbations and to assess whether any HRV group is more pathogenic. Methods: Viral throat swabs were taken on all adult exacerbations between Dec 2008 and May 2011. Positive HRV samples were stored and typed in more detail. Date matched non-CF samples positive for HRV were used as community controls. To allow typing the extracted viral RNA was transcribed into cDNA. The resulting cDNA underwent two rounds of nested PCR reaction to produce amplicons of the VP4/VP2 region. This was then sequenced to allow genetic typing of the specimens. The HRV groups were then compared using demographic and clinical data obtained from patient records. Conclusion: HRV was identified in 7% of pulmonary exacerbations. 126 positive samples were stored and typing was possible in 104 of the isolates. HRV group A was identied in 57 (55%) of isolates, group B in 23 (22%), and group C in 24 (23%). The frequency of HRV group A was higher than that seen in community (55% vs 40%). Some patients had viral persistence for several weeks with the same serotype. Virus associated exacerbations had significantly greater impact on lung function, length of treatment, and time to next exacerbation compared to matched patients without a virus. However, no individual HRV group appeared more pathogenic and the detrimental effects were seen with all groups.

Short Communication The non-primate hepacivirus 59 untranslated region possesses internal ribosomal entry site activity

Abstract: The 59 untranslated region (59UTR) of the recently described non-primate hepacivirus (NPHV) contains a region with sequence homology to the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) and GB virus B (GBV-B). Here, we demonstrated internal translation initiation by the NPHV 59UTR in a bicistronic vector. An RNA stem–loop upstream of the NPHV IRES was structurally distinct from corresponding regions in HCV and GBV-B, and was not required for IRES function. Insertion of the NPHV stem–loop into the corresponding region of the HCV 59UTR within the HCV subgenomic replicon significantly impaired RNA replication, indicating that longrange interactions between the 59UTR and cis-acting downstream elements within the NPHV genome are not interchangeable with those of HCV. Despite similarities in IRES structure and function between hepaciviruses, replication elements in the NPHV 59UTR appear functionally distinct from those of HCV.