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Factor VIII and IX clotting factor concentrates manufactured from pooled plasma have been identified as potent sources of virus infection in persons with hemophilia (PWHs) in the 1970s and 1980s. To investigate the range and diversity of viruses over this period, we analysed 24 clotting factor concentrates for several blood-borne viruses. Nucleic acid was extracted from 14 commercially produced clotting factors and 10 from nonremunerated donors, preserved in lyophilized form (expiry dates: 1974-1992). Clotting factors were tested by commercial and in-house quantitative PCRs for blood-borne viruses hepatitis A, B, C and E viruses (HAV, HBV, HCV, HEV), HIV- types 1/2, parvoviruses B19V and PARV4, and human pegiviruses types 1 and 2 (HPgV-1,-2). HCV and HPgV-1 were the most frequently detected viruses (both 14/24 tested) primarily in commercial clotting factors, with frequently extremely high viral loads in the late 1970s-1985 and a diverse range of HCV genotypes. Detection frequencies sharply declined following introduction of virus inactivation. HIV-1, HBV, and HAV were less frequently detected (3/24, 1/24, and 1/24 respectively); none were positive for HEV. Contrastingly, B19V and PARV4 were detected throughout the study period, even after introduction of dry heat treatment, consistent with ongoing documented transmission to PWHs into the early 1990s. While hemophilia treatment is now largely based on recombinant factor VIII/IX in the UK and elsewhere, the comprehensive screen of historical plasma-derived clotting factors reveals extensive exposure of PWHs to blood-borne viruses throughout 1970s-early 1990s, and the epidemiological and manufacturing parameters that influenced clotting factor contamination.
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Factores de Coagulación Sanguínea , Patógenos Transmitidos por la Sangre , Humanos , Patógenos Transmitidos por la Sangre/aislamiento & purificación , Infecciones de Transmisión Sanguínea/epidemiología , Infecciones de Transmisión Sanguínea/virología , Contaminación de Medicamentos , Historia del Siglo XX , Hemofilia A , Virus/clasificación , Virus/aislamiento & purificación , Virus/genética , Reacción en Cadena de la Polimerasa , Factor VIII , Factores de TiempoRESUMEN
The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models.
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COVID-19 , Inmunidad Innata , SARS-CoV-2 , Serina Endopeptidasas , Internalización del Virus , SARS-CoV-2/inmunología , SARS-CoV-2/fisiología , SARS-CoV-2/metabolismo , Humanos , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , COVID-19/virología , COVID-19/inmunología , COVID-19/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Replicación Viral , Animales , Endocitosis , Células HEK293 , Chlorocebus aethiops , CitologíaRESUMEN
Wastewater-based epidemiology (WBE) has emerged as an effective environmental surveillance tool for predicting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease outbreaks in high-income countries (HICs) with centralized sewage infrastructure. However, few studies have applied WBE alongside epidemic disease modelling to estimate the prevalence of SARS-CoV-2 in low-resource settings. This study aimed to explore the feasibility of collecting untreated wastewater samples from rural and urban catchment areas of Nagpur district, to detect and quantify SARS-CoV-2 using real-time qPCR, to compare geographic differences in viral loads, and to integrate the wastewater data into a modified Susceptible-Exposed-Infectious-Confirmed Positives-Recovered (SEIPR) model. Of the 983 wastewater samples analyzed for SARS-CoV-2 RNA, we detected significantly higher sample positivity rates, 43.7% (95% confidence interval (CI) 40.1, 47.4) and 30.4% (95% CI 24.66, 36.66), and higher viral loads for the urban compared with rural samples, respectively. The Basic reproductive number, R0, positively correlated with population density and negatively correlated with humidity, a proxy for rainfall and dilution of waste in the sewers. The SEIPR model estimated the rate of unreported coronavirus disease 2019 (COVID-19) cases at the start of the wave as 13.97 [95% CI (10.17, 17.0)] times that of confirmed cases, representing a material difference in cases and healthcare resource burden. Wastewater surveillance might prove to be a more reliable way to prepare for surges in COVID-19 cases during future waves for authorities.
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COVID-19 , SARS-CoV-2 , Aguas Residuales , India/epidemiología , COVID-19/epidemiología , COVID-19/virología , COVID-19/diagnóstico , Humanos , Aguas Residuales/virología , SARS-CoV-2/aislamiento & purificación , Carga Viral , Pandemias , Monitoreo Epidemiológico Basado en Aguas Residuales , Aguas del Alcantarillado/virologíaRESUMEN
BACKGROUND: Coronavirus disease 2019 (COVID-19) was officially declared a pandemic by the World Health Organisation (WHO) on 11 March 2020, as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread rapidly across the world. We investigated the seroprevalence of anti-SARS-CoV-2 antibodies in pediatric patients on dialysis or kidney transplantation in the UK. METHODS: Excess sera samples were obtained prospectively during outpatient visits or haemodialysis sessions and analysed using a custom immunoassay calibrated with population age-matched healthy controls. Two large pediatric centres contributed samples. RESULTS: In total, 520 sera from 145 patients (16 peritoneal dialysis, 16 haemodialysis, 113 transplantation) were analysed cross-sectionally from January 2020 until August 2021. No anti-SARS-CoV-2 antibody positive samples were detected in 2020 when lockdown and enhanced social distancing measures were enacted. Thereafter, the proportion of positive samples increased from 5% (January 2021) to 32% (August 2021) following the emergence of the Alpha variant. Taking all patients, 32/145 (22%) were seropositive, including 8/32 (25%) with prior laboratory-confirmed SARS-CoV-2 infection and 12/32 (38%) post-vaccination (one of whom was also infected after vaccination). The remaining 13 (41%) seropositive patients had no known stimulus, representing subclinical cases. Antibody binding signals were comparable across patient ages and dialysis versus transplantation and highest against full-length spike protein versus spike subunit-1 and nucleocapsid protein. CONCLUSIONS: Anti-SARS-CoV-2 seroprevalence was low in 2020 and increased in early 2021. Serological surveillance complements nucleic acid detection and antigen testing to build a greater picture of the epidemiology of COVID-19 and is therefore important to guide public health responses. A higher resolution version of the Graphical abstract is available as Supplementary information.
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COVID-19 , Trasplante de Riñón , Humanos , Niño , Trasplante de Riñón/efectos adversos , SARS-CoV-2 , Diálisis Renal/efectos adversos , COVID-19/epidemiología , Estudios Seroepidemiológicos , Control de Enfermedades Transmisibles , Anticuerpos Antivirales , Reino Unido/epidemiologíaRESUMEN
Background: The COVID-19 pandemic showcased the power of genomic sequencing to tackle the emergence and spread of infectious diseases. However, metagenomic sequencing of total microbial RNAs in wastewater has the potential to assess multiple infectious diseases simultaneously and has yet to be explored. Methods: A retrospective RNA-Seq epidemiological survey of 140 untreated composite wastewater samples was performed across urban (n = 112) and rural (n = 28) areas of Nagpur, Central India. Composite wastewater samples were prepared by pooling 422 individual grab samples collected prospectively from sewer lines of urban municipality zones and open drains of rural areas from 3rd February to 3rd April 2021, during the second COVID-19 wave in India. Samples were pre-processed and total RNA was extracted prior to genomic sequencing. Findings: This is the first study that has utilised culture and/or probe-independent unbiased RNA-Seq to examine Indian wastewater samples. Our findings reveal the detection of zoonotic viruses including chikungunya, Jingmen tick and rabies viruses, which have not previously been reported in wastewater. SARS-CoV-2 was detectable in 83 locations (59%), with stark abundance variations observed between sampling sites. Hepatitis C virus was the most frequently detected infectious virus, identified in 113 locations and co-occurring 77 times with SARS-CoV-2; and both were more abundantly detected in rural areas than urban zones. Concurrent identification of segmented virus genomic fragments of influenza A virus, norovirus, and rotavirus was observed. Geographical differences were also observed for astrovirus, saffold virus, husavirus, and aichi virus that were more prevalent in urban samples, while the zoonotic viruses chikungunya and rabies, were more abundant in rural environments. Interpretation: RNA-Seq can effectively detect multiple infectious diseases simultaneously, facilitating geographical and epidemiological surveys of endemic viruses that could help direct healthcare interventions against emergent and pre-existent infectious diseases as well as cost-effectively and qualitatively characterising the health status of the population over time. Funding: UK Research and Innovation (UKRI) Global Challenges Research Fund (GCRF) grant number H54810, as supported by Research England.
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BACKGROUND AND AIMS: The newly developed direct-acting antivirals have revolutionized the treatment of chronic hepatitis C virus (HCV), with cure rates as high as 98% in some cohorts. Although genome sequencing has demonstrated that some subtypes of HCV naturally harbor drug resistance associated substitutions (RAS), these are often overlooked as "rarities." Furthermore, commercial subtyping assays and associated epidemiological findings are skewed towards Western cohorts and whole-genome sequencing can be problematic to deploy without significant infrastructure and training support. We thus aimed to develop a simple, robust and accurate HCV subtyping pipeline, to optimize and streamline molecular detection and sequence-based typing of diverse RAS-containing subtypes. METHODS: HCV serum derived from 146 individuals, whose likely source of infection was from sub-Saharan Africa (SSA) was investigated with a novel panel of single round polymerase chain reaction (PCR) assays targeting NS5B and NS5A genomic regions. Virus subtype assignments were determined by pairwise-distance analysis and compared to both diagnostic laboratory assignments and free-to-use online typing tools. RESULTS: Partial NS5A and NS5B sequences were respectively obtained from 131 to 135 HCV-positive patients born in 19 different countries from SSA but attending clinics in the UK. We determined that routine clinical diagnostic methods incorrectly subtyped 59.0% of samples, with a further 6.8% incorrectly genotyped. Of five commonly used online tools, Geno2Pheno performed most effectively in determining a subtype in agreement with pairwise distance analysis. CONCLUSION: This study provides a simple low-cost pathway to accurately subtype in SSA, guide regional therapeutic choice and assist global surveillance and elimination initiatives.
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Hepatitis C Crónica , Hepatitis C , Humanos , Hepatitis C Crónica/diagnóstico , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/epidemiología , Antivirales/uso terapéutico , Antivirales/farmacología , Proteínas no Estructurales Virales/genética , Hepatitis C/diagnóstico , Hepatitis C/tratamiento farmacológico , Hepatitis C/epidemiología , Hepacivirus/genética , Genotipo , África del Sur del Sahara/epidemiología , Reino Unido/epidemiología , Farmacorresistencia Viral/genéticaRESUMEN
Analysis of host genetic polymorphisms is an increasingly important tool for understanding and predicting pathogenesis and treatment response of viral diseases. The gene locus of scavenger receptor class B type I (SR-BI), encoding a cell entry factor and receptor for hepatitis C virus (HCV), contains several genetic polymorphisms. We applied a probe extension assay to determine the frequency of six single nucleotide polymorphisms (SNPs) within the SR-BI gene locus in 374 individuals with history of HCV infection. In addition, SR-BI messenger RNA (mRNA) levels were analyzed in liver biopsy specimens of chronically infected HCV subjects. The rs5888 variant allele T was present at a higher frequency in subjects with advanced fibrosis (χ2 , p = 0.016) and after adjusting for age, duration of infection and alcohol intake as confounding factors. Haplotype analysis of SNP frequencies showed that a haplotype consisting of rs61932577 variant allele C and rs5888 variant allele T was associated with an increased risk of advanced liver fibrosis (defined by an Ishak score 4-6) (adjusted odds ratio 2.81; 95% confidence interval 1.06-7.46. p = 0.038). Carriers of the rs5888 variant allele T displayed reduced SR-BI mRNA expression in liver biopsy specimens. In conclusion the rs5888 polymorphism variant is associated with decreased SR-BI expression and an increased risk of development of advanced fibrosis in chronic HCV infection. These findings provide further evidence for a role of SR-BI in HCV pathogenesis and provides a genetic marker for prediction of those infected individuals at greater risk of developing severe disease.
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Hepatitis C Crónica , Receptores Depuradores de Clase B , Humanos , Hepacivirus/metabolismo , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/genética , Gravedad del Paciente , ARN Mensajero/metabolismo , Receptores Depuradores de Clase B/metabolismoRESUMEN
A better understanding of the antibody response during natural infection and the effect on disease progression and reinfection is necessary for the development of a protective hepatitis C virus (HCV) vaccine. The HCV pseudoparticle (HCVpp) system enables the study of viral entry and inhibition by antibody neutralization. A robust and comparable neutralization assay is crucial for the development and evaluation of experimental vaccines.With the aim of optimizing the HCVpp-murine leukaemia virus (MLV) system, we tested the neutralization of HCVpp-harbouring E1E2 from 21 HCV isolates representing 6 different genotypes by several monoclonal antibodies (mAbs). HCVpps are generated by expressing functional envelope glycoproteins (E1E2) onto pseudoparticles derived from env-deleted MLV. Adjustments of E1E2, gag-pol and luciferase plasmid ratios resulted in increased yields for most HCVpps and recovery of one non-infectious HCVpp. We simplified and improved the protocol to achieve higher signal/noise ratios and minimized the amount of HCVpps and mAbs needed for the detection of neutralization. Using our optimized protocol, we demonstrated comparable results to previously reported data with both diluted and freeze-thawed HCVpps.In conclusion, we successfully established a simplified and reproducible HCVpp neutralization protocol for studying a wide range of HCV variants. This simplified protocol provides highly consistent results and could be easily adopted by others to evaluate precious biological material. This will contribute to a better understanding of the antibody response during natural infection and help evaluate experimental HCV vaccines.
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Hepatitis C , Vacunas , Animales , Ratones , Hepacivirus , Anticuerpos Neutralizantes , Anticuerpos contra la Hepatitis C , Pruebas de Neutralización , Proteínas del Envoltorio Viral/genética , Hepatitis C/genética , Anticuerpos MonoclonalesRESUMEN
Hepatitis C virus (HCV) is responsible for more than 180 million infections worldwide, and about 80â% of infections are reported in Low and Middle-income countries (LMICs). Therapy is based on the administration of interferon (INF), ribavirin (RBV) or more recently Direct-Acting Antivirals (DAAs). However, amino acid substitutions associated with resistance (RAS) have been extensively described and can contribute to treatment failure, and diagnosis of RAS requires considerable infrastructure, not always locally available. Dried serum spots (DSS) sampling is an alternative specimen collection method, which embeds drops of serum onto filter paper to be transported by posting to a centralized laboratory. Here, we assessed feasibility of genotypic analysis of HCV from DSS in a cohort of 80 patients from São Paulo state Brazil. HCV RNA was detected on DSS specimens in 83â% of samples of HCV infected patients. HCV genotypes 1a, 1b, 2a, 2c and 3a were determined using the sequence of the palm domain of NS5B region, and RAS C316N/Y, Q309R and V321I were identified in HCV 1b samples. Concerning therapy outcome, 75â% of the patients who used INF +RBV as a previous protocol of treatment did not respond to DAAs, and 25â% were end-of-treatment responders. It suggests that therapy with INF plus RBV may contribute for non-response to a second therapeutic protocol with DAAs. One patient that presented RAS (V321I) was classified as non-responder, and combination of RAS C316N and Q309R does not necessarily imply in resistance to treatment in this cohort of patients. Data presented herein highlights the relevance of studying circulating variants for a better understanding of HCV variability and resistance to the therapy. Furthermore, the feasibility of carrying out genotyping and RAS phenotyping analysis by using DSS card for the potential of informing future treatment interventions could be relevant to overcome the limitations of processing samples in several location worldwide, especially in LMICs.
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BACKGROUND: Human Parainfluenza viruses (HPIV) comprise of four members of the genetically distinct genera of Respirovirus (HPIV1&3) and Orthorubulavirus (HPIV2&4), causing significant upper and lower respiratory tract infections worldwide, particularly in children. However, despite frequent molecular diagnosis, they are frequently considered collectively or with HPIV4 overlooked entirely. We therefore investigated clinical and viral epidemiological distinctions of the relatively less prevalent Orthorubulaviruses HPIV2&4 at a regional UK hospital across four autumn/winter epidemic seasons. METHODS: A retrospective audit of clinical features of all HPIV2 or HPIV4 RT-PCR-positive patients, diagnosed between 1st September 2013 and 12th April 2017 was undertaken, alongside sequencing of viral genome fragments in a representative subset of samples. RESULTS: Infection was observed across all age groups, but predominantly in children under nine and adults over 40, with almost twice as many HPIV4 as HPIV2 cases. Fever, abnormal haematology, elevated C-reactive protein and hospital admission were more frequently seen in HPIV2 than HPIV4 infection. Each of the four seasonal peaks of either HPIV2, HPIV4 or both, closely matched that of RSV, occurring in November and December and preceding that of Influenza A. A subset of viruses were partially sequenced, indicating co-circulation of multiple subtypes of both HPIV2&4, but with little variation between each epidemic season or from limited global reference sequences. CONCLUSIONS: Despite being closest known genetic relatives, our data indicates a potential difference in associated disease between HPIV2 and HPIV4, with more hospitalisation seen in HPIV2 mono-infected individuals, but a greater overall number of HPIV4 cases.
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Infecciones por Paramyxoviridae , Infecciones del Sistema Respiratorio , Adulto , Proteína C-Reactiva , Niño , Genómica , Humanos , Epidemiología Molecular , Virus de la Parainfluenza 1 Humana/genética , Virus de la Parainfluenza 2 Humana/genética , Virus de la Parainfluenza 3 Humana/genética , Infecciones por Paramyxoviridae/diagnóstico , Infecciones por Paramyxoviridae/epidemiología , Infecciones del Sistema Respiratorio/epidemiología , Estudios Retrospectivos , Reino Unido/epidemiologíaRESUMEN
Enterovirus D68 (EV-D68) has recently been identified in biennial epidemics coinciding with diagnoses of non-polio acute flaccid paralysis/myelitis (AFP/AFM). We investigated the prevalence, genetic relatedness and associated clinical features of EV-D68 in 193 EV-positive samples from 193 patients in late 2018, UK. EV-D68 was detected in 83 (58â%) of 143 confirmed EV-positive samples. Sequencing and phylogenetic analysis revealed extensive genetic diversity, split between subclades B3 (n=50) and D1 (n=33), suggesting epidemiologically unrelated infections. B3 predominated in children and younger adults, and D1 in older adults and the elderly (P=0.0009). Clinical presentation indicated causation or exacerbation of respiratory distress in 91.4â% of EV-D68-positive individuals, principally cough (75.3â%), shortness of breath (56.8â%), coryza (48.1â%), wheeze (46.9â%), supplemental oxygen required (46.9â%) and fever (38.9â%). Two cases of AFM were observed, one with EV-D68 detectable in the cerebrospinal fluid, but otherwise neurological symptoms were rarely reported (n=4). Both AFM cases and all additional instances of intensive care unit (ICU) admission (n=5) were seen in patients infected with EV-D68 subclade B3. However, due to the infrequency of severe infection in our cohort, statistical significance could not be assessed.
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Enterovirus Humano D , Infecciones por Enterovirus , Epidemias , Anciano , Enfermedades Virales del Sistema Nervioso Central , Niño , Enterovirus Humano D/genética , Infecciones por Enterovirus/diagnóstico , Infecciones por Enterovirus/epidemiología , Variación Genética , Humanos , Mielitis , Enfermedades Neuromusculares , Filogenia , Reino Unido/epidemiologíaRESUMEN
Following acute HCV infection, the virus establishes a chronic disease in the majority of patients whilst few individuals clear the infection spontaneously. The precise mechanisms that determine chronic HCV infection or spontaneous clearance are not completely understood but are proposed to be driven by host and viral genetic factors as well as HCV encoded immunomodulatory proteins. Using the HIV-1 LTR as a tool to measure NF-κB activity, we identified that the HCV E1E2 glycoproteins and more so the E2 protein down-modulates HIV-1 LTR activation in 293T, TZM-bl and the more physiologically relevant Huh7 liver derived cell line. We demonstrate this effect is specifically mediated through inhibiting NF-κB binding to the LTR and show that this effect was conserved for all HCV genotypes tested. Transcriptomic analysis of 293T cells expressing the HCV glycoproteins identified E1E2 mediated stimulation of the endoplasmic reticulum (ER) stress response pathway and upregulation of stress response genes such as ATF3. Through shRNA mediated inhibition of ATF3, one of the components, we observed that E1E2 mediated inhibitory effects on HIV-1 LTR activity was alleviated. Our in vitro studies demonstrate that HCV Env glycoprotein activates host ER Stress Pathways known to inhibit NF-κB activity. This has potential implications for understanding HCV induced immune activation as well as oncogenesis.
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Hepatitis C , FN-kappa B , Estrés del Retículo Endoplásmico , Glicoproteínas , Humanos , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
Hepatitis C virus (HCV) infection affects more than 71 million people worldwide. The disease slowly progresses to chronic, long-term liver injury which leads to hepatocellular carcinoma (HCC) in 5â% of infections. The alternative reading frame protein (ARFP/core+1) is encoded by a sequence overlapping the HCV core gene in the +1 reading frame. Its role in hepatitis C pathogenesis and the viral life cycle is unclear, although some observers have related its production to disease progression and the development of HCC. The aim of this study was to determine whether ARFP is immunogenic in patients with chronic HCV genotype 3 infection and to assess whether sero-reactivity is associated with disease progression, particularly to HCC. Immunogenic epitopes within the protein were predicted by a bioinformatics tool, and three -20 aa length-peptides (ARFP-P1, ARFP-P2 and ARFP-P3) were synthesized and used in an avidin-biotin ARFP/core+1 peptide ELISA. Serum samples from 50 patients with chronic HCV genotype 3 infection, 50 genotype-1 patients, 50 HBV patients and 110 healthy controls were tested. Sero-reactivity to the ARFP peptides was also tested and compared in 114 chronic HCV genotype-3 patients subdivided on the basis of disease severity into non-cirrhotic, cirrhotic and HCC groups. Chronic HCV genotype-3 patients showed noticeable rates of reactivity to ARFP and core peptides. Seropositivity rates were 58% for ARFP-P1, 47â% for ARFP-P2, 5.9â% for ARFP-P3 and 100â% for C22 peptides. There was no significant difference between these seroreactivities between HCV genotype-3 patients with HCC, and HCV genotype-3 patients with and without liver cirrhosis. Patients with chronic HCV genotype-3 infection frequently produce antibodies against ARFP/core+1 protein. ARFP peptide reactivity was not associated with disease severity in patients with HCV genotype-3. These results support the conclusion that ARFP/core+1 is produced during HCV infection, but they do not confirm that antibodies to ARFP can indicate HCV disease progression.
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Carcinoma Hepatocelular , Hepatitis C Crónica , Hepatitis C , Neoplasias Hepáticas , Progresión de la Enfermedad , Genotipo , Hepacivirus , Anticuerpos contra la Hepatitis C , Humanos , Péptidos/genética , Sistemas de Lectura , Proteínas del Núcleo Viral/metabolismoRESUMEN
BACKGROUND: Specialized proresolution molecules (SPMs) halt the transition to chronic pathogenic inflammation. We aimed to quantify serum levels of pro- and anti-inflammatory bioactive lipids in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients, and to identify potential relationships with innate responses and clinical outcome. METHODS: Serum from 50 hospital admitted inpatients (22 female, 28 male) with confirmed symptomatic SARS-CoV-2 infection and 94 age- and sex-matched controls collected prior to the pandemic (SARS-CoV-2 negative), were processed for quantification of bioactive lipids and anti-nucleocapsid and anti-spike quantitative binding assays. RESULTS: SARS-CoV-2 serum had significantly higher concentrations of omega-6-derived proinflammatory lipids and omega-6- and omega-3-derived SPMs, compared to the age- and sex-matched SARS-CoV-2-negative group, which were not markedly altered by age or sex. There were significant positive correlations between SPMs, proinflammatory bioactive lipids, and anti-spike antibody binding. Levels of some SPMs were significantly higher in patients with an anti-spike antibody value >0.5. Levels of linoleic acid and 5,6-dihydroxy-8Z,11Z,14Z-eicosatrienoic acid were significantly lower in SARS-CoV-2 patients who died. CONCLUSIONS: SARS-CoV-2 infection was associated with increased levels of SPMs and other pro- and anti-inflammatory bioactive lipids, supporting the future investigation of the underlying enzymatic pathways, which may inform the development of novel treatments.
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COVID-19 , SARS-CoV-2 , Inmunidad Adaptativa , Anticuerpos Antivirales , Eicosanoides , Femenino , Humanos , Masculino , Glicoproteína de la Espiga del CoronavirusRESUMEN
BACKGROUND & AIMS: Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays. METHODS: We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps. RESULTS: Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones. CONCLUSIONS: Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.
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Variación Antigénica/inmunología , Antígenos Virales/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Hepacivirus/inmunología , Pruebas de Neutralización/métodos , Proteínas del Envoltorio Viral/inmunología , Variación Antigénica/genética , Antígenos Virales/genética , Línea Celular Tumoral , Hepacivirus/genética , Hepatitis C/prevención & control , Humanos , Inmunogenicidad Vacunal , Reproducibilidad de los Resultados , Desarrollo de Vacunas , Proteínas del Envoltorio Viral/genética , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
Understanding the impact of prior infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the response to vaccination is a priority for responding to the coronavirus disease 2019 (COVID-19) pandemic. In particular, it is necessary to understand how prior infection plus vaccination can modulate immune responses against variants of concern. To address this, we sampled 20 individuals with and 25 individuals without confirmed previous SARS-CoV-2 infection from a large cohort of health care workers followed serologically since April 2020. All 45 individuals had received two doses of the Pfizer-BioNTech BNT162b2 vaccine with a delayed booster at 10 weeks. Absolute and neutralizing antibody titers against wild-type SARS-CoV-2 and variants were measured using enzyme immunoassays and pseudotype neutralization assays. We observed antibody reactivity against lineage A, B.1.351, and P.1 variants with increasing antigenic exposure, through either vaccination or natural infection. This improvement was further confirmed in neutralization assays using fixed dilutions of serum samples. The impact of antigenic exposure was more evident in enzyme immunoassays measuring SARS-CoV-2 spike proteinspecific IgG antibody concentrations. Our data show that multiple exposures to SARS-CoV-2 spike protein in the context of a delayed booster expand the neutralizing breadth of the antibody response to neutralization-resistant SARS-CoV-2 variants. This suggests that additional vaccine boosts may be beneficial in improving immune responses against future SARS-CoV-2 variants of concern.
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COVID-19 , SARS-CoV-2 , Formación de Anticuerpos , Vacuna BNT162 , Vacunas contra la COVID-19 , HumanosRESUMEN
Although SARS-CoV-2 vaccines are very safe, we report 4 cases of the bifacial weakness with paresthesias variant of Guillain-Barré syndrome (GBS) occurring within 3 weeks of vaccination with the Oxford-AstraZeneca SARS-CoV-2 vaccine. This rare neurological syndrome has previously been reported in association with SARS-CoV-2 infection itself. Our cases were given either intravenous immunoglobulin, oral steroids, or no treatment. We suggest vigilance for cases of bifacial weakness with paresthesias variant GBS following vaccination for SARS-CoV-2 and that postvaccination surveillance programs ensure robust data capture of this outcome, to assess for causality. ANN NEUROL 2021;90:315-318.
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Vacunas contra la COVID-19/efectos adversos , Síndrome de Guillain-Barré/inducido químicamente , Síndrome de Guillain-Barré/diagnóstico , Vacunas contra la COVID-19/administración & dosificación , Glucocorticoides/administración & dosificación , Síndrome de Guillain-Barré/tratamiento farmacológico , Humanos , Inmunoglobulinas Intravenosas/administración & dosificación , Masculino , Persona de Mediana Edad , Prednisolona/administración & dosificación , Vacunación/efectos adversos , Adulto JovenRESUMEN
Hepatitis C virus (HCV) has no animal reservoir, infecting only humans. To investigate species barrier determinants limiting infection of rodents, murine liver complementary DNA library screening was performed, identifying transmembrane proteins Cd302 and Cr1l as potent restrictors of HCV propagation. Combined ectopic expression in human hepatoma cells impeded HCV uptake and cooperatively mediated transcriptional dysregulation of a noncanonical program of immunity genes. Murine hepatocyte expression of both factors was constitutive and not interferon inducible, while differences in liver expression and the ability to restrict HCV were observed between the murine orthologs and their human counterparts. Genetic ablation of endogenous Cd302 expression in human HCV entry factor transgenic mice increased hepatocyte permissiveness for an adapted HCV strain and dysregulated expression of metabolic process and host defense genes. These findings highlight human-mouse differences in liver-intrinsic antiviral immunity and facilitate the development of next-generation murine models for preclinical testing of HCV vaccine candidates.
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Hepacivirus , Hepatitis C , Animales , Hepacivirus/genética , Ratones , Ratones Transgénicos , Internalización del VirusRESUMEN
Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or 'viroporins', contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery. We describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.
Asunto(s)
Antivirales/farmacología , Hepacivirus/metabolismo , Proteínas Virales/antagonistas & inhibidores , Animales , Antivirales/química , Biomarcadores , Línea Celular , Perros , Descubrimiento de Drogas , Genotipo , Hepacivirus/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Conformación Proteica , Relación Estructura-Actividad , Proteínas Virales/metabolismoRESUMEN
BACKGROUND: Human enteroviruses (EV) are the leading cause of viral meningitis. EV genotyping is predominantly performed through amplification and sequencing of viral capsid protein-1 (VP1), frequently by national reference laboratories (NRLs). OBJECTIVE: To determine the frequency of genotyping failure in our NRL-submitted samples and apply a superior alternative assay to resolve untyped specimens. STUDY DESIGN: We initially audited genotyping data received for a cohort of patients in the East Midlands, UK by the NRL between 2013 and 2017, then identified an alternative RT-PCR typing method by literature review and evaluated primers from both assays in silico against comprehensive publicly available genomic data. The alternative assay was further optimised and applied to archived nucleic acids from previously untypable samples. RESULTS: Genotyping data showed a significant increase in untypable EV strains through the study period (pâ¯=â¯0.0073). Typing failure appeared unrelated to sample type or viral load. In silico analyses of 2,201â¯EV genomes showed high levels of mismatch between reference assay primers and clinically significant EV-species, in contrast to a selected alternative semi-nested RT-PCR VP1-typing assay. This alternative assay, with minor modifications, successfully genotyped 23 of 24 previously untypable yet viable archived specimens (EV-A, nâ¯=â¯4; EV-B, nâ¯=â¯19). Phylogenetic analyses identified no predominant strain within NRL untypable isolates, suggesting sub-optimal reference assay sensitivity across EV species, in agreement with in silico analyses. CONCLUSION: This modified highly sensitive RT-PCR assay presents a suitable alternative to the current English national reference VP1-typing assay and is recommended in other settings experiencing typing failure.