RESUMEN
Most human pegivirus 2 (HPgV-2) infections are associated with past or current hepatitis C virus (HCV) infection. HPgV-2 is thought to be a bloodborne virus: higher prevalence of active infection has been found in populations with a history of parenteral exposure to viruses. We evaluated longitudinally collected blood samples obtained from injection drug users (IDUs) for active and resolved HPgV-2 infections using a combination of HPgV-2-specific molecular and serologic tests. We found evidence of HPgV-2 infection in 11.2% (22/197) of past or current HCV-infected IDUs, compared with 1.9% (4/205) of an HCV-negative IDU population. Testing of available longitudinal blood samples from HPgV-2-positive participants identified 5 with chronic infection (>6 months viremia in >3 timepoints); 2 were identified among the HCV-positive IDUs and 3 among the HCV-negative IDUs. Our findings indicate that HPgV-2 can establish chronic infection and replicate in the absence of HCV.
Asunto(s)
Consumidores de Drogas , Infecciones por Flaviviridae/epidemiología , Hepatitis C , Pegivirus/aislamiento & purificación , Adolescente , Adulto , California/epidemiología , Coinfección , Femenino , Infecciones por Flaviviridae/sangre , Infecciones por Flaviviridae/virología , Humanos , Estudios Longitudinales , Masculino , Prevalencia , Asunción de Riesgos , Encuestas y Cuestionarios , Adulto JovenRESUMEN
Hepatitis C virus (HCV) seroconversion among HCV-uninfected transplant recipients from HCV-infected (NAT+/Antibody+) or HCV-exposed (NAT-/Antibody+) donors has been reported. However, the origin of anti-HCV antibody and the implications of seroconversion remain unknown. We longitudinally tested plasma from HCV-uninfected kidney (n = 31) or heart transplant recipients (n = 9) of an HCV NAT+ organ for anti-HCV antibody (both IgG and IgM isotypes). Almost half of all participants had detectable anti-HCV antibody at any point during follow-up. The majority of antibody-positive individuals became positive within 1-3 days of transplantation, and 6 recipients had detectable antibody on the first day posttransplant. Notably, all anti-HCV antibody was IgG, even in samples collected posttransplant day 1. Late seroconversion was uncommon (≈20%-25% of antibody+ recipients). Early antibody persisted over 30 days in kidney recipients, whereas early antibody dropped below detection in 50% of heart recipients within 2 weeks after transplant. Anti-HCV antibody is common in HCV-uninfected recipients of an HCV NAT+ organ. The IgG isotype of this antibody and the kinetics of its appearance and durability suggest that anti-HCV antibody is donor derived and is likely produced by a cellular source. Our data suggest that transfer of donor humoral immunity to a recipient may be much more common than previously appreciated.
Asunto(s)
Insuficiencia Cardíaca/cirugía , Trasplante de Corazón/efectos adversos , Anticuerpos contra la Hepatitis C/sangre , Hepatitis C/transmisión , Fallo Renal Crónico/cirugía , Trasplante de Riñón/efectos adversos , Anciano , Femenino , Insuficiencia Cardíaca/complicaciones , Insuficiencia Cardíaca/virología , Hepacivirus , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Terapia de Inmunosupresión , Fallo Renal Crónico/complicaciones , Fallo Renal Crónico/virología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Factores de Riesgo , Obtención de Tejidos y Órganos , Receptores de Trasplantes , Carga ViralRESUMEN
The prevalence of chronic hepatitis C virus (HCV) and the presence of human pegivirus 2 (HPgV-2) have not been examined in Cameroon, although HCV has been associated with HPgV-2 infections previously. Herein we aimed to characterize the burden and genetic diversity of HCV and the presence of HPgV-2 in Cameroon. Retrospective plasma specimens collected from N = 12 369 consenting subjects in South Cameroon from 2013 to 2016 were included in the study. The majority (97.1%) of participants were patients seeking health care. All specimens were screened for HCV using the Abbott RealTime HCV viral load assay and positive specimens with remaining volume were also screened for HPgV-2 antibodies on the Abbott ARCHITECT instrument, followed by molecular characterization. Overall, HCV RNA was detected in 305 (2.47%; 95% CI: 2.21%-2.75%) specimens. Notably, the prevalence of HCV RNA was 9.09% amongst participants over age 40 and 3.81% amongst males. Phylogenetic classification of N = 103 HCV sequences identified genotypes 1 (19.4%), 2 (15.5%) and 4 (65.1%) within the study cohort. Amongst HCV RNA-positive specimens, N = 28 (10.6%; 95% CI: 7.44%-14.90%) specimens also had detectable HPgV-2 antibodies. Of these, N = 2 viremic HPgV-2 infections were confirmed by sequencing and shared 93-94 median % identity with strains found on other continents. This is the first study to determine the prevalence of chronic HCV in Cameroon, and the discovery of HPgV-2 in this study cohort expands the geography of HPgV-2 to the African continent, indicating a widespread distribution exists.
Asunto(s)
Anticuerpos Antivirales/sangre , Monitoreo Epidemiológico , Infecciones por Flaviviridae/epidemiología , Flaviviridae/aislamiento & purificación , Hepatitis C Crónica/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Camerún/epidemiología , Niño , Preescolar , Coinfección/epidemiología , Coinfección/virología , Femenino , Flaviviridae/genética , Infecciones por Flaviviridae/sangre , Hepacivirus/genética , Hepatitis C Crónica/sangre , Humanos , Lactante , Masculino , Persona de Mediana Edad , Filogenia , Prevalencia , ARN Viral/sangre , Estudios Retrospectivos , Adulto JovenRESUMEN
The tick-borne protozoan Babesia microti is responsible for more than 200 cases of transfusion-transmitted babesiosis (TTB) infection in the United States that have occurred over the last 30 years. Measures to mitigate the risk of TTB include nucleic acid testing (NAT) and B. microti antibody testing. A fully automated prototype B. microti antibody test was developed on the Architect instrument. The specificity was determined to be 99.98% in volunteer blood donors (n = 28,740) from areas considered to have low endemicity for B. microti The sensitivity of the prototype test was studied in experimentally infected macaques; a total of 128 samples were detected as positive whereas 125 were detected as positive with an indirect fluorescent antibody (IFA) test; additionally, 83 (89.2%) of the PCR-positive samples were detected in contrast to 81 (87.1%) using an IFA test. All PCR-positive samples that tested negative in the prototype antibody test were preseroconversion period samples. Following seroconversion, periods of intermittent parasitemia occurred; 17 PCR-negative samples drawn in between PCR-positive bleed dates tested positive both by the prototype test (robust reactivity) and IFA test (marginal reactivity) prior to the administration of therapeutic drugs, indicating that the PCR test failed to detect samples from persistently infected macaques. The prototype assay detected 56 of 58 (96.6%) human subjects diagnosed with clinical babesiosis by both PCR and IFA testing. Overall, the prototype anti-Babesia assay provides a highly sensitive and specific test for the diagnosis of B. microti infection. While PCR is preferred for detection of window-period parasitemia, antibody tests detect infected subjects during periods of low-level parasitemia.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Babesia microti/aislamiento & purificación , Babesiosis/diagnóstico , Inmunoensayo/normas , Parasitemia/diagnóstico , Animales , Anticuerpos Antiprotozoarios/inmunología , Babesia microti/genética , Babesia microti/inmunología , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente Indirecta/normas , Humanos , Inmunoensayo/instrumentación , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Macaca , Tamizaje Masivo , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Seroconversión , Reacción a la Transfusión/prevención & controlRESUMEN
A novel blood-borne human pegivirus (HPgV), HPgV-2, was recently identified in hepatitis C virus (HCV)-infected individuals and individuals who had received multiple transfusions. Robust serological assays capable of detecting antibodies in HPgV-2-infected individuals are needed to establish global seroprevalence rates and potential disease associations. The two objectives of this study were to determine the utility of mammalian cell-expressed HPgV-2 E2 glycoprotein or bacterium-expressed nonstructural protein 4AB (NS4AB) in detecting past or present infections and to compare the total prevalence (antibody and RNA positive) of HPgV-2 with that of the other human pegivirus, HPgV-1 (GB virus C [GBV-C]). HPgV-2 E2 antibodies were detected in 13 (92.86%) of 14 HPgV-2-viremic cases, and NS4AB antibodies were detected in 8 (57.14%) of 14 cases. The HPgV-2 seroprevalence was significantly higher (P < 0.0001) among HCV-infected individuals (3.31% [24 of 726 samples]) than among non-HCV-infected individuals (0.30% [4 of 1,348 samples]). Of 31 anti-E2-positive samples, 22 had supplemental supporting data; 12 samples were HPgV-2 RNA positive and 10 nonviremic samples were antibody positive for peptides or NS4AB. The total prevalence of HPgV-1 (35.00%) was significantly higher than that of HPgV-2 (1.33%) in all populations tested (P < 0.0001). For HPgV-1, codetection of antibodies to E2 and RNA was infrequent (5.88%). In contrast, antibodies to E2 were detected in most HPgV-2-viremic individuals (92.86%), as is observed among individuals chronically infected with HCV, most of whom are antibody positive for HCV E2. Our studies indicate that HPgV-2 circulates with HCV and displays a profile similar to the serological profile of HCV-infected persons, although the pathogenicity of this virus has yet to be established.
Asunto(s)
Anticuerpos Antivirales/sangre , Infecciones por Flaviviridae/epidemiología , Infecciones por Flaviviridae/virología , Flaviviridae/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , Infecciones por Flaviviridae/inmunología , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Estudios SeroepidemiológicosRESUMEN
The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells.
Asunto(s)
Apolipoproteínas E , Retículo Endoplásmico , Aparato de Golgi , Hepacivirus/fisiología , Vesículas Secretoras , Proteína 1 de Membrana Asociada a Vesículas , Liberación del Virus/fisiología , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Transporte Biológico Activo/genética , Línea Celular , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Aparato de Golgi/virología , Humanos , Vesículas Secretoras/genética , Vesículas Secretoras/metabolismo , Vesículas Secretoras/virología , Proteína 1 de Membrana Asociada a Vesículas/genética , Proteína 1 de Membrana Asociada a Vesículas/metabolismo , Ensamble de Virus/fisiologíaRESUMEN
Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets.
Asunto(s)
Endocitosis/fisiología , Genes Virales/genética , Hepacivirus/genética , Hepacivirus/metabolismo , Interferencia de ARN , ARN/análisis , Animales , Técnica del Anticuerpo Fluorescente , Biblioteca de Genes , Humanos , Reacción en Cadena de la Polimerasa , Proteínas Virales/genética , Proteínas Virales/metabolismo , Acoplamiento Viral , Internalización del VirusRESUMEN
There is an urgent need for innovative methods to reduce transmission of bloodborne pathogens like HIV and HCV among people who inject drugs (PWID). We investigate if PWID who acquire non-pathogenic bloodborne viruses like anelloviruses and pegiviruses might be at greater risk of acquiring a bloodborne pathogen. PWID who later acquire HCV accumulate more non-pathogenic viruses in plasma than matched controls who do not acquire HCV infection. Additionally, phylogenetic analysis of those non-pathogenic virus sequences reveals drug use networks. Here we find first in Baltimore and confirm in San Francisco that the accumulation of non-pathogenic viruses in PWID is a harbinger for subsequent acquisition of pathogenic viruses, knowledge that may guide the prioritization of the public health resources to combat HIV and HCV.
Asunto(s)
Infecciones de Transmisión Sanguínea , Plasma , Trastornos Relacionados con Sustancias , Viroma , Adulto , Secuencia de Aminoácidos , Anelloviridae , Patógenos Transmitidos por la Sangre , Femenino , Hepatitis C/epidemiología , Humanos , Conocimiento , Masculino , Metagenómica , Filogenia , Salud Pública , Adulto JovenRESUMEN
Axonal transport is essential for the successful establishment of neuroinvasive herpesvirus infections in peripheral ganglia (retrograde transport) and the subsequent spread to exposed body surfaces following reactivation from latency (anterograde transport). We examined two components of pseudorabies virus (US3 and UL13), both of which are protein kinases, as potential regulators of axon transport. Following replication of mutant viruses lacking kinase activity, newly assembled capsids displayed an increase in retrograde motion that prevented efficient delivery of capsids to the distal axon. The aberrant increase in retrograde motion was accompanied by loss of a viral membrane marker from the transported capsids, indicating that the viral kinases allow for efficient anterograde transport by stabilizing membrane-capsid interactions during the long transit from the neuron cell body to the distal axon.
Asunto(s)
Transporte Axonal , Axones/virología , Cápside/enzimología , Herpesviridae , Proteínas Quinasas/metabolismo , Células Receptoras Sensoriales/virología , Animales , Axones/metabolismo , Línea Celular , Embrión de Pollo , Células Epiteliales/virología , Herpesviridae/enzimología , Herpesviridae/genética , Herpesviridae/patogenicidad , Mutagénesis , Proteínas Quinasas/genética , Transporte de Proteínas , PorcinosRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Hepatitis C virus (HCV) enters hepatocytes via various entry factors, including scavenger receptor BI (SR-B1), cluster of differentiation 81 (CD81), epidermal growth factor receptor (EGFR), claudin-1 (CLDN1), and occludin (OCLN). As CLDN1 and OCLN are not readily accessible due to their tight junctional localization, HCV likely accesses them by either disrupting cellular polarity or migrating to the tight junction. In this study, we image HCV entry into a three-dimensional polarized hepatoma system and reveal that the virus sequentially engages these entry factors through actin-dependent mechanisms. HCV initially localizes with the early entry factors SR-B1, CD81, and EGFR at the basolateral membrane and then accumulates at the tight junction in an actin-dependent manner. HCV associates with CLDN1 and then OCLN at the tight junction and is internalized via clathrin-mediated endocytosis by an active process requiring EGFR. Thus, HCV uses a dynamic and multi-step process to engage and enter host cells.
Asunto(s)
Carcinoma Hepatocelular/diagnóstico por imagen , Carcinoma Hepatocelular/virología , Hepacivirus/fisiología , Hepatitis C/virología , Imagenología Tridimensional/métodos , Organoides/diagnóstico por imagen , Organoides/metabolismo , Organoides/virología , Internalización del Virus , Actinas/metabolismo , Técnicas de Cultivo de Célula/métodos , Línea Celular , Membrana Celular/metabolismo , Supervivencia Celular , Claudina-1/metabolismo , Endocitosis/fisiología , Receptores ErbB/metabolismo , Hepacivirus/patogenicidad , Interacciones Huésped-Patógeno/fisiología , Humanos , Ocludina/metabolismo , Receptores Depuradores de Clase B/metabolismo , Tetraspanina 28/metabolismo , Uniones Estrechas/metabolismo , Proteínas no Estructurales Virales/metabolismoRESUMEN
Hepatitis E virus (HEV) is a viral pathogen transmitted by the fecal-oral route and is a major cause of waterborne acute hepatitis in many developing countries. In addition to infecting humans, HEV has been identified in swine, wild boars, rabbits and other mammals; with swine and wild boars being main reservoirs for zoonotic transmission of HEV. There are four major HEV genotypes known to infect humans; genotypes 1 (HEV-1) and 2 (HEV-2) are restricted to humans, and genotypes 3 (HEV-3) and 4 (HEV-4) are zoonotic. Herein, three human HEV strains originating in France were sequenced and near full-length genomes were characterized. Phylogenetic analysis showed that two strains were genotype 3 and closely grouped (a 100% bootstrap value) with subtype 3i reference strains. In percent nucleotide identities, these two strains were 94% identical to each other, 90-93% identical to subtype 3i strains, 82-86% identical to other HEV-3, and 77-79% identical to rabbit HEV strains excluding the two divergent strains KJ013414 and KJ013415 (74%); these two strains were less than 77% identical to strains of HEV genotypes 1, 2 and 4. The third strain was found distinct from any known HEV strains in the database, and located between the clusters of HEV-3 and rabbit HEV strains. This unique strain was 74-75% identical to HEV-1, 73% to HEV-2, 81-82% to HEV-3, 77-79% to rabbit HEV again excluding the two divergent strains KJ013414 and KJ013415 (74%), and 74-75% to HEV-4, suggesting a novel unclassified strain associated with HEV-3 and rabbit HEV. SimPlot and BootScan analyses revealed a putative recombination of HEV-3 and rabbit HEV sequences at four breakpoints. Phylogenetic trees of the five fragments of the genome confirmed the presence of two HEV-3 derived and three unclassified sequences. Analyses of the amino acid sequences of the three open reading frames (ORF1-3) encoded proteins of these three novel strains showed that some amino acid residues specific to rabbit HEV strains were found solely in this unclassified strain but not in the two newly identified genotype 3i strains. The results obtained by SimPlots, BootScans, phylogenetic analyses, and amino acid sequence comparisons in this study all together appear to suggest that this novel unclassified strain is likely carrying a mosaic genome derived from HEV-3 and rabbit HEV sequences, and is thus designated as a putative genotype 3/rabbit HEV recombinant.
Asunto(s)
Genoma Viral/genética , Virus de la Hepatitis E/genética , Animales , Genotipo , Humanos , Sistemas de Lectura Abierta/genética , ARN Viral/genética , Conejos , Análisis de Secuencia de ADN , PorcinosRESUMEN
Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), infects an estimated 15-20 million people worldwide and confers a greater risk for accelerated progression to liver disease. However, limited HDV surveillance data are available in sub-Saharan Africa where HDV diversity is high. To determine the prevalence and diversity of HDV in Cameroon, serological and molecular characterization was performed on 1928 HBsAg positive specimens selected from retrospective viral surveillance studies conducted in Cameroon from 2010-2016. Samples were screened for HDV antibodies on the Abbott ARCHITECT instrument and for HDV RNA on the Abbott m2000 instrument by research assays. HDV positive specimens with sufficient viral load were selected for genomic sequencing. The seroprevalence of HDV in HBsAg positive samples from Cameroon was 46.73% [95% CI; 44.51-48.96%], with prevalence of active HDV infection being 34.2% [95% CI; 32.09-36.41%]. HDV genotypes 1, 6, 7 and 8 were identified amongst N = 211 sequences, including N = 145 genomes. HDV prevalence is high within the study cohort, indicating that a large portion of HBV infected individuals in Cameroon are at elevated risk for severe hepatitis and death. Collectively, these results emphasize the need for HBV vaccination and HDV testing in HBsAg positive patients in Cameroon.
Asunto(s)
Genoma Viral , Hepatitis D , Virus de la Hepatitis Delta , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Camerún/epidemiología , Niño , Preescolar , Femenino , Anticuerpos Antihepatitis/sangre , Hepatitis D/sangre , Hepatitis D/epidemiología , Hepatitis D/genética , Virus de la Hepatitis Delta/genética , Virus de la Hepatitis Delta/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Estudios SeroepidemiológicosRESUMEN
Worldwide, an estimated 5% of hepatitis B virus (HBV) infected people are coinfected with hepatitis delta virus (HDV). HDV infection leads to increased mortality over HBV mono-infection, yet HDV diagnostics are not widely available. Prototype molecular (RNA) and serologic (IgG) assays were developed for high-throughput testing on the Abbott m2000 and ARCHITECT systems, respectively. RNA detection was achieved through amplification of a ribozyme region target, with a limit of detection of 5 IU/ml. The prototype serology assay (IgG) was developed using peptides derived from HDV large antigen (HDAg), and linear epitopes were further identified by peptide scan. Specificity of an HBV negative population was 100% for both assays. A panel of 145 HBsAg positive samples from Cameroon with unknown HDV status was tested using both assays: 16 (11.0%) had detectable HDV RNA, and 23 (15.7%) were sero-positive including the 16 HDV RNA positive samples. Additionally, an archival serial bleed panel from an HDV superinfected chimpanzee was tested with both prototypes; data was consistent with historic testing data using a commercial total anti-Delta test. Overall, the two prototype assays provide sensitive and specific methods for HDV detection using high throughput automated platforms, allowing opportunity for improved diagnosis of HDV infected patients.
Asunto(s)
Anticuerpos Antivirales/sangre , Hepatitis B/diagnóstico , Virus de la Hepatitis Delta/fisiología , Antígenos de Hepatitis delta/sangre , ARN Viral/genética , Pruebas Serológicas/métodos , Animales , Hepatitis B/sangre , Hepatitis B/virología , Antígenos de Hepatitis delta/inmunología , Pan troglodytes , SeroconversiónRESUMEN
Human Pegivirus 2 (HPgV-2) was recently identified in the bloodstream of HCV-infected and multiply transfused individuals. Initial reports show HPgV-2 circulates at a low prevalence in HCV co-infected individuals, necessitating testing of large cohorts of samples to identify infected persons. The identification of additional HPgV-2 cases was facilitated by the development of a high throughput and reliable molecular reverse transcription polymerase chain reaction (RT-PCR) assay intended for use on the automated Abbott m2000 system with a capability of extracting and testing 96 samples at once. A dual target approach was taken to reduce the risk of a false-negative result, amplifying sequences within the 5' UTR and NS2/3 coding regions of HPgV-2. The assay was expanded to multiplex detection of the other human Pegivirus, HPgV-1 (formerly GBV-C), to allow simultaneous prevalence comparison. The limit of detection (LOD; 95% detection) for HPgV-2 was experimentally determined to be 126 copies/mL. Through use of the newly developed multiplex assay, 21 strains of HPgV-2 circulating in HCV past or present infections were identified, with all strains confirmed by next generation sequencing. The multiplexed assay has high specificity and showed no cross-reactivity of HPgV-2 with HPgV-1 or other Flaviviruses. This automated assay will be instrumental in future studies addressing HPgV-2 pathogenicity, prevalence, and sequence diversity.
Asunto(s)
Virus GB-C/aislamiento & purificación , Reacción en Cadena de la Polimerasa Multiplex/métodos , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Regiones no Traducidas 5' , Automatización de Laboratorios , Coinfección/virología , Infecciones por Flaviviridae/virología , Virus GB-C/clasificación , Virus GB-C/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Límite de Detección , Filogenia , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Proteínas no Estructurales Virales/genéticaRESUMEN
Different viruses exploit the host cytoskeleton to facilitate replication and spread. The conserved US3 protein of the alphaherpesvirus pseudorabies virus induces actin stress fiber disassembly and formation of actin-containing cell projections, which are associated with enhanced intercellular virus spread. Proteins of members of other virus families, notably vaccinia virus F11L protein and human immunodeficiency virus Nef protein, induce actin rearrangements that are very similar to those induced by US3. Interestingly, unlike F11L and Nef, the US3 protein displays serine/threonine kinase activity. Here, we report that the kinase activity of pseudorabies virus US3 is absolutely required for its actin modulating activity. These data show that different viruses have developed independent mechanisms to induce very similar actin rearrangements.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Herpesvirus Suido 1/enzimología , Herpesvirus Suido 1/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Seudorrabia/patología , Proteínas Virales/metabolismo , Animales , Línea Celular , Citoesqueleto/enzimología , RatonesRESUMEN
Upon entering a cell, alphaherpesvirus capsids are transported toward the minus ends of microtubules and ultimately deposit virus DNA within the host nucleus. The virus proteins that mediate this centripetal transport are unknown but are expected to be either viral tegument proteins, which are a group of capsid-associated proteins, or a surface component of the capsid itself. Starting with derivatives of pseudorabies virus that encode a fluorescent protein fused to a structural component of the virus, we have made a collection of 12 mutant viruses that lack either the VP26 capsid protein or an individual tegument protein. Using live-cell fluorescence microscopy, we tracked individual virus particles in axons following infection of primary sensory neurons. Quantitative analysis of the VP26-null virus indicates that this protein plays no observable role in capsid transport. Furthermore, viruses lacking tegument proteins that are nonessential for virus propagation in cell culture were also competent for axonal transport. These results indicate that a protein essential for viral propagation mediates transport of the capsid to the nucleus.