Geographical variation in hepatitis C-related severe liver disease and patient risk factors: a multicentre cross-sectional study.

Despite promising steps towards the elimination of hepatitis C virus (HCV) in the UK, several indicators provide a cause for concern for future disease burden. We aimed to improve understanding of geographical variation in HCV-related severe liver disease and historic risk factor prevalence among clinic attendees in England and Scotland. We used metadata from 3829 HCV-positive patients consecutively enrolled into HCV Research UK from 48 hospital centres in England and Scotland during 2012-2014. Employing mixed-effects statistical modelling, several independent risk factors were identified: age 46-59 y (ORadj 3.06) and ≥60 y (ORadj 5.64) relative to <46 y, male relative to female sex (ORadj 1.58), high BMI (ORadj 1.73) and obesity (ORadj 2.81) relative to normal BMI, diabetes relative to no diabetes (ORadj 2.75), infection with HCV genotype (GT)-3 relative to GT-1 (ORadj 1.75), route of infection through blood products relative to injecting drug use (ORadj 1.40), and lower odds were associated with black ethnicity (ORadj 0.31) relative to white ethnicity. A small proportion of unexplained variation was attributed to differences between hospital centres and local health authorities. Our study provides a baseline measure of historic risk factor prevalence and potential geographical variation in healthcare provision, to support ongoing monitoring of HCV-related disease burden and the design of risk prevention measures.

Determining the cellular diversity of hepatitis C virus quasispecies by single-cell viral sequencing.

Single-cell genomics is emerging as an important tool in cellular biology. We describe for the first time a system to investigate RNA virus quasispecies diversity at the cellular level utilizing hepatitis C virus (HCV) replicons. A high-fidelity nested reverse transcription (RT)-PCR assay was developed, and validation using control transcripts of known copy number indicated a detection limit of 3 copies of viral RNA/reaction. This system was used to determine the cellular diversity of subgenomic JFH-1 HCV replicons constitutively expressed in Huh7 cells. Each cell contained a unique quasispecies that was much less diverse than the quasispecies of the bulk cell population from which the single cells were derived, suggesting the occurrence of independent evolution at the cellular level. An assessment of the replicative fitness of the predominant single-cell quasispecies variants indicated a modest reduction in fitness compared to the wild type. Real-time RT-PCR methods capable of determining single-cell viral loads were developed and indicated an average of 113 copies of replicon RNA per cell, correlating with calculated RNA copy numbers in the bulk cell population. This study introduces a single-cell RNA viral-sequencing method with numerous potential applications to explore host-virus interactions during infection. HCV quasispecies diversity varied greatly between cells in vitro, suggesting different within-cell evolutionary pathways. Such divergent trajectories in vivo could have implications for the evolution and establishment of antiviral-resistant variants and host immune escape mutants.

Development and assay of RNA transcripts of enterovirus species A to D, rhinovirus species a to C, and human parechovirus: assessment of assay sensitivity and specificity of real-time screening and typing methods.

Nucleic acid amplification methods such as the PCR have had a major impact on the diagnosis of viral infections, often achieving greater sensitivities and shorter turnaround times than conventional assays and an ability to detect viruses refractory to conventional isolation methods. Their effectiveness is, however, significantly influenced by assay target sequence variability due to natural diversity and rapid sequence changes in viruses that prevent effective binding of primers and probes. This was investigated for a diverse range of enteroviruses (EVs; species A to D), human rhinoviruses (HRVs; species A to C), and human parechovirus (HPeV) in a multicenter assay evaluation using a series of full-length prequantified RNA transcripts. RNA concentrations were quantified by absorption (NanoDrop) and fluorescence methods (RiboGreen) prior to dilution in buffer supplemented with RNase inhibitors and carrier RNA. RNA transcripts were extremely stable, showing minimal degradation after prolonged storage at temperatures between ambient and -20°C and after multiple freeze-thaw cycles. Transcript dilutions distributed to six referral laboratories were screened by real-time reverse transcriptase PCR assays using different primers and probes. All of the laboratories reported high assay sensitivities for EV and HPeV transcripts approaching single copies and similar amplification kinetics for all four EV species. HRV detection sensitivities were more variable, often with substantially impaired detection of HRV species C. This could be accounted for in part by the placement of primers and probes to genetically variable target regions. Transcripts developed in this study provide reagents for the ongoing development of effective diagnostics that accommodate increasing knowledge of genetic heterogeneity of diagnostic targets.

Rapid simultaneous detection of enterovirus and parechovirus RNAs in clinical samples by one-step real-time reverse transcription-PCR assay.

Enteroviruses (EVs) are recognized as the major etiological agent in meningitis in children and young adults. The use of molecular techniques, such as PCR, has substantially improved the sensitivity of enterovirus detection compared to that of virus culture methods. PCR-based methods also can detect a much wider range of EV variants, including those within species A, as well as human parechoviruses (HPeVs) that often grow poorly in vitro and which previously have been underdiagnosed by traditional methods. To exploit these developments, we developed a real-time one-step reverse transcription-PCR (RT-PCR) for the rapid and sensitive detection of EV and HPeV in clinical specimens. Two commercially available RT-PCR kits were used (method I, Platinum one-step kit; method II, Express qPCR one-step kit) with primers and probes targeting the EV and HPeV 5'-untranslated regions (5'UTR). Amplification dynamics (threshold cycle [C(T)]values and efficiencies) of absolutely quantified full-length RNA transcripts representative of EV species A to D and HPeV were similar, demonstrating the effectiveness of both assays across the range of currently described human EV and HPeV variants. Probit analysis of multiple endpoint replicates demonstrated comparable sensitivities of the assays for EV and HPeV (method I, approximately 10 copies per reaction for both targets; method II, 20 copies per reaction). C(T) values were highly reproducible on repeat testing of positive controls within assays and between assay runs. Considering the sample turnaround time of less than 3 h, the multiplexed one-step RT-PCR method provides rapid diagnostic testing for EV and HPeV in cases of suspected central nervous system infections in a clinically relevant time frame.

Analysis of genetic diversity and sites of recombination in human rhinovirus species C.

Human rhinoviruses (HRVs) are a highly prevalent and diverse group of respiratory viruses. Although HRV-A and HRV-B are traditionally detected by virus isolation, a series of unculturable HRV variants have recently been described and assigned as a new species (HRV-C) within the picornavirus Enterovirus genus. To investigate their genetic diversity and occurrence of recombination, we have performed comprehensive phylogenetic analysis of sequences from the 5' untranslated region (5' UTR), VP4/VP2, VP1, and 3Dpol regions amplified from 89 HRV-C-positive respiratory samples and available published sequences. Branching orders of VP4/VP2, VP1, and 3Dpol trees were identical, consistent with the absence of intraspecies recombination in the coding regions. However, numerous tree topology changes were apparent in the 5' UTR, where >60% of analyzed HRV-C variants showed recombination with species A sequences. Two recombination hot spots in stem-loop 5 and the polypyrimidine tract in the 5' UTR were mapped using the program GroupingScan. Available HRV-C sequences showed evidence for additional interspecies recombination with HRV-A in the 2A gene, with breakpoints mapping precisely to the boundaries of the C-terminal domain of the encoded proteinase. Pairwise distances between HRV-C variants in VP1 and VP4/VP2 regions fell into two separate distributions, resembling inter- and intraserotype distances of species A and B. These observations suggest that, without serological cross-neutralization data, HRV-C genetic groups may be equivalently classified into types using divergence thresholds derived from distance distributions. The extensive sequence data from multiple genome regions of HRV-C and analyses of recombination in the current study will assist future formulation of consensus criteria for HRV-C type assignment and identification.

Comparison of human parechovirus and enterovirus detection frequencies in cerebrospinal fluid samples collected over a 5-year period in edinburgh: HPeV type 3 identified as the most common picornavirus type.

Human enteroviruses (EVs) and more recently parechoviruses (HPeVs) have been identified as the principal viral causes of neonatal sepsis-like disease and meningitis. The relative frequencies of specific EV and HPeV types were determined over a 5-year surveillance period using highly sensitive EV and HPeV PCR assays for screening 4,168 cerebrospinal fluid (CSF) specimens collected from hospitalized individuals between 2005 and 2010 in Edinburgh. Positive CSF samples were typed by sequencing of VP1. From the 201 EV and 31 HPeV positive (uncultured) CSF samples on screening, a high proportion of available samples could be directly typed (176/182, 97%). Highest frequencies of EV infections occurred in young adults (n = 43; 8.6%) although a remarkably high proportion of positive samples (n = 98; 46%) were obtained from young infants (<3 months). HPeV infections were seen exclusively in children under the age of 3 months (31/1,105; 2.8%), and confined to spring on even-numbered years (22% in March 2006, 25% in April 2008, and 22% in March 2010). In contrast, EV infections were distributed widely across the years. Twenty different EV serotypes were detected; E9, E6, and CAV9 being found most frequently, whereas all but one HPeVs were type 3. Over this period, HPeV3 was identified as the most prevalent picornavirus type in CNS-related infections with similarly high incidences of EV infection frequencies in very young children. The highly sensitive virus typing methods applied in this study will assist further EV and HPeV screening of sepsis and meningitis cases as well as in future molecular epidemiological studies and population surveillance.

Genomic epidemiology reveals multiple introductions of SARS-CoV-2 from mainland Europe into Scotland.

Coronavirus disease 2019 (COVID-19) was first diagnosed in Scotland on 1 March 2020. During the first month of the outbreak, 2,641 cases of COVID-19 led to 1,832 hospital admissions, 207 intensive care admissions and 126 deaths. We aimed to identify the source and number of introductions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into Scotland using a combined phylogenetic and epidemiological approach. Sequencing of 1,314 SARS-CoV-2 viral genomes from available patient samples enabled us to estimate that SARS-CoV-2 was introduced to Scotland on at least 283 occasions during February and March 2020. Epidemiological analysis confirmed that early introductions of SARS-CoV-2 originated from mainland Europe (the majority from Italy and Spain). We identified subsequent early outbreaks in the community, within healthcare facilities and at an international conference. Community transmission occurred after 2 March, 3 weeks before control measures were introduced. Earlier travel restrictions or quarantine measures, both locally and internationally, would have reduced the number of COVID-19 cases in Scotland. The risk of multiple reintroduction events in future waves of infection remains high in the absence of population immunity.

Incidence, molecular epidemiology and clinical presentations of human metapneumovirus; assessment of its importance as a diagnostic screening target.

BACKGROUND: Human metapneumovirus (HMPV) is a recently discovered human paramyxovirus associated with a spectrum of respiratory symptoms from the common cold to pneumonia and bronchiolitis. OBJECTIVES: To assess the clinical significance and epidemiology of HMPV, standardized comparison of frequencies of infection, age profiles and disease associations were made with other respiratory viruses in Scotland. STUDY DESIGN: 7091 respiratory samples collected in Scotland between 1 July 2006 and 30 June 2008 from 4282 individuals were screened by multiplex RT-PCR for respiratory syncytial virus (HRSV), adenovirus (AdV), parainfluenza viruses 1-3 (PIV-1, -2 and -3), influenza A and B and by nested RT-PCR for HMPV. RESULTS: HMPV was the fifth most prevalent virus (2.0% of samples), found predominantly in young children in winter months. In the 2006-2007 respiratory season, 70% of HMPV isolates were genotype A, but a switch to predominantly type B infections occurred next winter. For samples with information on clinical presentations, 26% of HMPV infections were from subjects with lower respiratory tract presentations, lower than recorded for HRSV, but similar to adenovirus, parainfluenza viruses and influenza viruses A and B. Around 13% of HMPV infections were associated with upper respiratory tract symptoms or disease, comparable with other respiratory virus infections. CONCLUSIONS: Numerically and through its association with respiratory disease, HMPV represents a diagnostically significant target that should be included in PCR-based routine screening of respiratory samples. Understanding the biological basis of observed rapid turnover of HMPV variants, including the observed HMPV genotype change between respiratory seasons requires further longitudinal studies.

Genetics, recombination and clinical features of human rhinovirus species C (HRV-C) infections; interactions of HRV-C with other respiratory viruses.

To estimate the frequency, molecular epidemiological and clinical associations of infection with the newly described species C variants of human rhinoviruses (HRV), 3243 diagnostic respiratory samples referred for diagnostic testing in Edinburgh were screened using a VP4-encoding region-based selective polymerase chain reaction (PCR) for HRV-C along with parallel PCR testing for 13 other respiratory viruses. HRV-C was the third most frequently detected behind respiratory syncytial virus (RSV) and adenovirus, with 141 infection episodes detected among 1885 subjects over 13 months (7.5%). Infections predominantly targeted the very young (median age 6-12 months; 80% of infections in those <2 years), occurred throughout the year but with peak incidence in early winter months. HRV-C was detected significantly more frequently among subjects with lower (LRT) and upper respiratory tract (URT) disease than controls without respiratory symptoms; HRV-C mono-infections were the second most frequently detected virus (behind RSV) in both disease presentations (6.9% and 7.8% of all cases respectively). HRV variants were classified by VP4/VP2 sequencing into 39 genotypically defined types, increasing the current total worldwide to 60. Through sequence comparisons of the 5'untranslated region (5'UTR), the majority grouped with species A (n = 96; 68%, described as HRV-Ca), the remainder forming a phylogenetically distinct 5'UTR group (HRV-Cc). Multiple and bidirectional recombination events between HRV-Ca and HRV-Cc variants and with HRV species A represents the most parsimonious explanation for their interspersed phylogeny relationships in the VP4/VP2-encoding region. No difference in age distribution, seasonality or disease associations was identified between HRV-Ca and HRV-Cc variants. HRV-C-infected subjects showed markedly reduced detection frequencies of RSV and other respiratory viruses, providing evidence for a major interfering effect of HRV-C on susceptibility to other respiratory virus infections. HRV-C's disease associations, its prevalence and evidence for interfering effects on other respiratory viruses mandates incorporation of rhinoviruses into future diagnostic virology screening.

pH and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon.

The effects of changes in the gut environment upon the human colonic microbiota are poorly understood. The response of human fecal microbial communities from two donors to alterations in pH (5.5 or 6.5) and peptides (0.6 or 0.1%) was studied here in anaerobic continuous cultures supplied with a mixed carbohydrate source. Final butyrate concentrations were markedly higher at pH 5.5 (0.6% peptide mean, 24.9 mM; 0.1% peptide mean, 13.8 mM) than at pH 6.5 (0.6% peptide mean, 5.3 mM; 0.1% peptide mean, 7.6 mM). At pH 5.5 and 0.6% peptide input, a high butyrate production coincided with decreasing acetate concentrations. The highest propionate concentrations (mean, 20.6 mM) occurred at pH 6.5 and 0.6% peptide input. In parallel, major bacterial groups were monitored by using fluorescence in situ hybridization with a panel of specific 16S rRNA probes. Bacteroides levels increased from ca. 20 to 75% of total eubacteria after a shift from pH 5.5 to 6.5, at 0.6% peptide, coinciding with high propionate formation. Conversely, populations of the butyrate-producing Roseburia group were highest (11 to 19%) at pH 5.5 but fell at pH 6.5, a finding that correlates with butyrate formation. When tested in batch culture, three Bacteroides species grew well at pH 6.7 but poorly at pH 5.5, which is consistent with the behavior observed for the mixed community. Two Roseburia isolates grew equally well at pH 6.7 and 5.5. These findings suggest that a lowering of pH resulting from substrate fermentation in the colon may boost butyrate production and populations of butyrate-producing bacteria, while at the same time curtailing the growth of Bacteroides spp.