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1.
Genome Res ; 2024 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-39299904

RESUMEN

Variant detection from long-read genome sequencing (lrGS) has proven to be more accurate and comprehensive than variant detection from short-read genome sequencing (srGS). However, the rate at which lrGS can increase molecular diagnostic yield for rare disease is not yet precisely characterized. We performed lrGS using Pacific Biosciences HiFi technology on 96 short-read-negative probands with rare diseases that were suspected to be genetic. We generated hg38-aligned variants and de novo phased genome assemblies, and subsequently annotated, filtered, and curated variants using clinical standards. New disease-relevant or potentially relevant genetic findings were identified in 16/96 (16.7%) probands, nine of which (8/96, ~9.4%) harbored pathogenic or likely pathogenic variants. Nine probands (~9.4%) had variants that were accurately called in both srGS and lrGS and represent changes to clinical interpretation, mostly from recently published gene-disease associations. Seven cases included variants that were only correctly interpreted in lrGS, including copy-number variants, an inversion, a mobile element insertion, two low-complexity repeat expansions, and a 1 bp deletion. While evidence for each of these variants is, in retrospect, visible in srGS, they were either not called within srGS data, were represented by calls with incorrect sizes or structures, or failed quality-control and filtration. Thus, while reanalysis of older srGS data clearly increases diagnostic yield, we find that lrGS allows for substantial additional yield (7/96, 7.3%) beyond srGS. We anticipate that as lrGS analysis improves, and as lrGS datasets grow allowing for better variant frequency annotation, the additional lrGS-only rare disease yield will grow over time.

2.
Genet Med ; 25(8): 100884, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37161864

RESUMEN

PURPOSE: Neurodevelopmental disorders (NDDs) often result from rare genetic variation, but genomic testing yield for NDDs remains below 50%, suggesting that clinically relevant variants may be missed by standard analyses. Here, we analyze "poison exons" (PEs), which are evolutionarily conserved alternative exons often absent from standard gene annotations. Variants that alter PE inclusion can lead to loss of function and may be highly penetrant contributors to disease. METHODS: We curated published RNA sequencing data from developing mouse cortex to define 1937 conserved PE regions potentially relevant to NDDs, and we analyzed variants found by genome sequencing in multiple NDD cohorts. RESULTS: Across 2999 probands, we found 6 novel clinically relevant variants in PE regions. Five of these variants are in genes that are part of the sodium voltage-gated channel alpha subunit family (SCN1A, SCN2A, and SCN8A), which is associated with epilepsies. One variant is in SNRPB, associated with cerebrocostomandibular syndrome. These variants have moderate to high computational impact assessments, are absent from population variant databases, and in genes with gene-phenotype associations consistent with each probands reported features. CONCLUSION: With a very minimal increase in variant analysis burden (average of 0.77 variants per proband), annotation of PEs can improve diagnostic yield for NDDs and likely other congenital conditions.


Asunto(s)
Epilepsia , Animales , Ratones , Humanos , Exones/genética , Epilepsia/diagnóstico , Epilepsia/genética , Fenotipo , Secuencia de Bases , Genómica
3.
J Clin Microbiol ; 60(12): e0122722, 2022 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-36409098

RESUMEN

Laboratory confirmation of infection is an essential component of measles surveillance. Detection of measles-specific IgM in serum by enzyme-linked immunosorbent assay (ELISA) is the most common method used to confirm measles infection. ELISA formats vary, as does the sensitivity and specificity of each assay. Specimens collected within 3 days of rash onset can yield a false-negative result, which can delay confirmation of measles cases. Interfering substances can yield a false-positive result, leading to unnecessary public health interventions. The IgM capture assay developed at the Centers for Disease Control (CDC) was compared against five commercially available ELISA kits for the ability to detect measles virus-specific IgM in a panel of 90 well-characterized specimens. Serum samples were tested in triplicate using each commercial kit as recommended by the manufacturer. Using the CDC measles IgM capture assay as the reference test; the sensitivity and specificity for each commercial kit ranged from 50 to 83% and 86.9 to 98%, respectively. Discrepant results were observed for samples tested with all five commercial kits and ranged from 13.8 to 28.8% of the specimens tested. False-positive results occurred in 2.0 to 13.1% of sera, while negative results were observed in 16.7 to 50% of sera that were positive by the CDC measles IgM capture assay. Evaluation and interpretation of measles IgM serologic results can be complex, particularly in measles elimination settings. The performance characteristics of a measles IgM assay should be carefully considered when selecting an assay to achieve high-quality measles surveillance.


Asunto(s)
Sarampión , Humanos , Inmunoglobulina M , Ensayo de Inmunoadsorción Enzimática/métodos , Sarampión/diagnóstico , Sarampión/epidemiología , Virus del Sarampión , Sensibilidad y Especificidad , Anticuerpos Antivirales
4.
Genet Med ; 24(4): 851-861, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-34930662

RESUMEN

PURPOSE: SouthSeq is a translational research study that undertook genome sequencing (GS) for infants with symptoms suggestive of a genetic disorder. Recruitment targeted racial/ethnic minorities and rural, medically underserved areas in the Southeastern United States, which are historically underrepresented in genomic medicine research. METHODS: GS and analysis were performed for 367 infants to detect disease-causal variation concurrent with standard of care evaluation and testing. RESULTS: Definitive diagnostic (DD) or likely diagnostic (LD) genetic findings were identified in 30% of infants, and 14% of infants harbored an uncertain result. Only 43% of DD/LD findings were identified via concurrent clinical genetic testing, suggesting that GS testing is better for obtaining early genetic diagnosis. We also identified phenotypes that correlate with the likelihood of receiving a DD/LD finding, such as craniofacial, ophthalmologic, auditory, skin, and hair abnormalities. We did not observe any differences in diagnostic rates between racial/ethnic groups. CONCLUSION: We describe one of the largest-to-date GS cohorts of ill infants, enriched for African American and rural patients. Our results show the utility of GS because it provides early-in-life detection of clinically relevant genetic variations not detected by current clinical genetic testing, particularly for infants exhibiting certain phenotypic features.


Asunto(s)
Pruebas Diagnósticas de Rutina , Pruebas Genéticas , Secuencia de Bases , Mapeo Cromosómico , Pruebas Genéticas/métodos , Genómica , Humanos
5.
Immunity ; 38(4): 805-17, 2013 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-23583644

RESUMEN

CD4(+) T follicular helper (Tfh) cells provide the required signals to B cells for germinal center reactions that are necessary for long-lived antibody responses. However, it remains unclear whether there are CD4(+) memory T cells committed to the Tfh cell lineage after antigen clearance. By using adoptive transfer of antigen-specific memory CD4(+) T cell subpopulations in the lymphocytic choriomeningitis virus infection model, we found that there are distinct memory CD4(+) T cell populations with commitment to either Tfh- or Th1-cell lineages. Our conclusions are based on gene expression profiles, epigenetic studies, and phenotypic and functional analyses. Our findings indicate that CD4(+) memory T cells "remember" their previous effector lineage after antigen clearance, being poised to reacquire their lineage-specific effector functions upon antigen reencounter. These findings have important implications for rational vaccine design, where improving the generation and engagement of memory Tfh cells could be used to enhance vaccine-induced protective immunity.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Subgrupos de Linfocitos T/inmunología , Células TH1/inmunología , Traslado Adoptivo , Animales , Antígenos Virales/inmunología , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Metilación de ADN/inmunología , Epigénesis Genética/inmunología , Granzimas/genética , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores CXCR5/metabolismo , Transcriptoma
6.
J Clin Microbiol ; 58(6)2020 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-32238434

RESUMEN

Measurement of measles virus-specific IgG is used to assess presumptive evidence of immunity among immunocompetent individuals with uncertain immune or vaccination status. False-negative test results may lead to unnecessary quarantine and exclusion from activities such as employment, education, and travel or result in unnecessary revaccination. In contrast, false-positive results may fail to identify susceptible individuals and promote spread of disease by those who are exposed and unprotected. To better understand the performance characteristics of tests to detect measles IgG, we compared five widely used, commercially available measles IgG test platforms using a set of 223 well-characterized serum samples. Measles virus neutralizing antibodies were also measured by in vitro plaque reduction neutralization, the gold standard method, and compared to IgG test results. Discrepant results were observed for samples in the low-positive ranges of the most sensitive tests, but there was good agreement across platforms for IgG-negative sera and for samples with intermediate to high levels of IgG. False-negative test results occurred in approximately 11% of sera, which had low levels of neutralizing antibody.


Asunto(s)
Virus del Sarampión , Sarampión , Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoensayo , Inmunoglobulina G , Sarampión/diagnóstico , Pruebas de Neutralización , Sensibilidad y Especificidad
7.
Eur J Immunol ; 43(12): 3219-32, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24030473

RESUMEN

CD4(+) T follicular helper (TFH) cells are central for generation of long-term B-cell immunity. A defining phenotypic attribute of TFH cells is the expression of the chemokine R CXCR5, and TFH cells are typically identified by co-expression of CXCR5 together with other markers such as PD-1, ICOS, and Bcl-6. Herein, we report high-level expression of the nutrient transporter folate R 4 (FR4) on TFH cells in acute viral infection. Distinct from the expression profile of conventional TFH markers, FR4 was highly expressed by naive CD4(+) T cells, was downregulated after activation and subsequently re-expressed on TFH cells. Furthermore, FR4 expression was maintained, albeit at lower levels, on memory TFH cells. Comparative gene expression profiling of FR4(hi) versus FR4(lo) Ag-specific CD4(+) effector T cells revealed a molecular signature consistent with TFH and TH1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto-enzyme CD73, were enriched in TFH cells compared with TH1 cells, and phenotypic analysis confirmed expression of CD73 on TFH cells. As there is now considerable interest in developing vaccines that would induce optimal TFH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of TFH cells following vaccination and infection.


Asunto(s)
Regulación de la Expresión Génica/inmunología , Receptores de Superficie Celular/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , 5'-Nucleotidasa/biosíntesis , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/inmunología , Enfermedad Aguda , Animales , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Regulación de la Expresión Génica/genética , Proteína Coestimuladora de Linfocitos T Inducibles/biosíntesis , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Ratones , Ratones Transgénicos , Receptor de Muerte Celular Programada 1/biosíntesis , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Proteínas Proto-Oncogénicas c-bcl-6 , Receptores CXCR5/biosíntesis , Receptores CXCR5/genética , Receptores CXCR5/inmunología , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Virosis/genética , Virosis/inmunología , Virosis/metabolismo
8.
J Virol ; 87(13): 7737-46, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23637417

RESUMEN

Long-lived plasma cells that reside in the bone marrow constitutively produce antibody in the absence of antigen and are the cellular basis of durable humoral immunity. The generation of these long-lived plasma cells depends upon a series of highly orchestrated interactions between antigen-specific CD4 T cells and B cells and the formation of germinal centers (GCs). In this study, we have examined the role of the cytokine interleukin-21 (IL-21) in regulating humoral immunity during acute viral infections. Using IL-21 receptor-deficient (IL-21R(-/-)) mice, we found that virus-specific CD4 T cells were generated after infection with lymphocytic choriomeningitis virus (LCMV) and that these CD4 T cells differentiated into T follicular helper (TFH)-like cells in the absence of IL-21 signaling. There was also no defect in the formation of GCs, although after day 15 these GCs disappeared faster in IL-21R(-/-) mice than in wild-type mice. Isotype switching and the initial LCMV-specific IgG response were normal in IL-21R(-/-) mice. However, these mice exhibited a profound defect in generating long-lived plasma cells and in sustaining antibody levels over time. Similar results were seen after infection of IL-21R(-/-) mice with vesicular stomatitis virus and influenza virus. Using chimeric mice containing wild-type or IL-21R(-/-) CD4 T cells and B cells, we showed that both B and CD4 T cells need IL-21 signaling for generating long-term humoral immunity. Taken together, our results highlight the importance of IL-21 in humoral immunity to viruses.


Asunto(s)
Diferenciación Celular/inmunología , Inmunidad Humoral/inmunología , Interleucinas/inmunología , Células Plasmáticas/inmunología , Virosis/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Centro Germinal/inmunología , Pruebas de Hemaglutinación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pruebas de Neutralización , Células Plasmáticas/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Interleucina-21/genética
9.
medRxiv ; 2024 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-38585854

RESUMEN

Variant detection from long-read genome sequencing (lrGS) has proven to be considerably more accurate and comprehensive than variant detection from short-read genome sequencing (srGS). However, the rate at which lrGS can increase molecular diagnostic yield for rare disease is not yet precisely characterized. We performed lrGS using Pacific Biosciences "HiFi" technology on 96 short-read-negative probands with rare disease that were suspected to be genetic. We generated hg38-aligned variants and de novo phased genome assemblies, and subsequently annotated, filtered, and curated variants using clinical standards. New disease-relevant or potentially relevant genetic findings were identified in 16/96 (16.7%) probands, eight of which (8/96, 8.33%) harbored pathogenic or likely pathogenic variants. Newly identified variants were visible in both srGS and lrGS in nine probands (~9.4%) and resulted from changes to interpretation mostly from recent gene-disease association discoveries. Seven cases included variants that were only interpretable in lrGS, including copy-number variants, an inversion, a mobile element insertion, two low-complexity repeat expansions, and a 1 bp deletion. While evidence for each of these variants is, in retrospect, visible in srGS, they were either: not called within srGS data, were represented by calls with incorrect sizes or structures, or failed quality-control and filtration. Thus, while reanalysis of older data clearly increases diagnostic yield, we find that lrGS allows for substantial additional yield (7/96, 7.3%) beyond srGS. We anticipate that as lrGS analysis improves, and as lrGS datasets grow allowing for better variant frequency annotation, the additional lrGS-only rare disease yield will grow over time.

10.
bioRxiv ; 2023 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-36711854

RESUMEN

Purpose: Neurodevelopmental disorders (NDDs) often result from rare genetic variation, but genomic testing yield for NDDs remains around 50%, suggesting some clinically relevant rare variants may be missed by standard analyses. Here we analyze "poison exons" (PEs) which, while often absent from standard gene annotations, are alternative exons whose inclusion results in a premature termination codon. Variants that alter PE inclusion can lead to loss-of-function and may be highly penetrant contributors to disease. Methods: We curated published RNA-seq data from developing mouse cortex to define 1,937 PE regions conserved between humans and mice and potentially relevant to NDDs. We then analyzed variants found by genome sequencing in multiple NDD cohorts. Results: Across 2,999 probands, we found six clinically relevant variants in PE regions that were previously overlooked. Five of these variants are in genes that are part of the sodium voltage-gated channel alpha subunit family ( SCN1A, SCN2A , and SCN8A ), associated with epilepsies. One variant is in SNRPB , associated with Cerebrocostomandibular Syndrome. These variants have moderate to high computational impact assessments, are absent from population variant databases, and were observed in probands with features consistent with those reported for the associated gene. Conclusion: With only a minimal increase in variant analysis burden (most probands had zero or one candidate PE variants in a known NDD gene, with an average of 0.77 per proband), annotation of PEs can improve diagnostic yield for NDDs and likely other congenital conditions.

11.
J Pers Med ; 13(7)2023 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-37511639

RESUMEN

BACKGROUND: It is critical to understand the wide-ranging clinical and non-clinical effects of genome sequencing (GS) for parents in the NICU context. We assessed parents' experiences with GS as a first-line diagnostic tool for infants with suspected genetic conditions in the NICU. METHODS: Parents of newborns (N = 62) suspected of having a genetic condition were recruited across five hospitals in the southeast United States as part of the SouthSeq study. Semi-structured interviews (N = 78) were conducted after parents received their child's sequencing result (positive, negative, or variants of unknown significance). Thematic analysis was performed on all interviews. RESULTS: Key themes included that (1) GS in infancy is important for reproductive decision making, preparing for the child's future care, ending the diagnostic odyssey, and sharing results with care providers; (2) the timing of disclosure was acceptable for most parents, although many reported the NICU environment was overwhelming; and (3) parents deny that receiving GS results during infancy exacerbated parent-infant bonding, and reported variable impact on their feelings of guilt. CONCLUSION: Parents reported that GS during the neonatal period was useful because it provided a "backbone" for their child's care. Parents did not consistently endorse negative impacts like interference with parent-infant bonding.

13.
Antiviral Res ; 180: 104849, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32553844

RESUMEN

During the last decade multiple mumps outbreaks have occurred in the U.S. despite high two dose MMR coverage with most cases detected among two dose MMR vaccine recipients. Waning immunity, the evolution of wild-type virus strains, and settings with intense exposure have contributed to the resurgence of mumps. Typically, mumps virus infections resolve without serious clinical sequelae; however, serious complications may occur among unvaccinated or severely immunocompromised individuals. Favipiravir (T-705) has been shown to have in vitro anti-viral activity against a broad range of positive and negative strand RNA viruses. Here, we demonstrate that T-705 inhibits the growth of wildtype and vaccine strains of mumps virus in vitro at low micro-molar concentrations (EC50 8-10µM). We did not observe the development of resistance after five subsequent passages at low concentrations of drug. Both viral RNA and protein synthesis were selectively reduced compared to host mRNA and protein synthesis. Antiviral treatment options for mumps virus infection may be valuable, especially for areas with a high disease burden or for cases with severe complications. These results presented here suggest that further studies are warranted.


Asunto(s)
Amidas/farmacología , Antivirales/farmacología , Virus de la Parotiditis/efectos de los fármacos , Pirazinas/farmacología , Replicación Viral/efectos de los fármacos , Células A549 , Animales , Chlorocebus aethiops , Humanos , ARN Viral/antagonistas & inhibidores , Células Vero
14.
mSphere ; 5(6)2020 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-33208518

RESUMEN

Between 2015 and 2017, routine molecular surveillance in the United States detected multiple mumps viruses (MuVs) with mutations in the small hydrophobic (SH) gene compared to a reference virus of the same genotype. These mutations include an unusual pattern of uracil-to-cytosine hypermutations and other mutations resulting in the generation of premature stop codons or disruption of the canonical stop codon. The mumps virus SH protein may serve as a virulence factor, based on evidence that it inhibits apoptosis and innate immune signaling in vitro and that recombinant viruses that do not express the SH protein are attenuated in an animal model. In this study, mumps viruses bearing variant SH sequences were isolated from contemporary outbreak samples to evaluate the impact of the observed mutations on SH protein function. All isolates with variant SH sequences replicated in interferon-competent cells with no evidence of attenuation. Furthermore, all SH-variant viruses retained the ability to abrogate induction of NF-κB-mediated innate immune signaling in infected cells. Ectopic expression of variant mumps SH genes is consistent with findings from infection experiments, indicating that the observed abrogation of signaling was not mediated by other viral factors that may modulate innate immune signaling. Molecular surveillance is an important public health tool for monitoring the diversity of circulating mumps viruses and can provide insights into determinants of disease. These findings, in turn, will inform studies employing reverse genetics to elucidate the specific mechanisms of MuV pathogenesis and potential impacts of observed sequence variants on infectivity, fitness, and virulence.IMPORTANCE Mumps virus (MuV) outbreaks occur in the United States despite high coverage with measles, mumps, rubella (MMR) vaccine. Routine genotyping of laboratory-confirmed mumps cases has been practiced in the United States since 2006 to enhance mumps surveillance. This study reports the detection of unusual mutations in the small hydrophobic (SH) protein of contemporary laboratory-confirmed mumps cases and is the first to describe the impact of such mutations on SH protein function. These mutations are predicted to profoundly alter the amino acid sequence of the SH protein, which has been shown to antagonize host innate immune responses; however, they were neither associated with defects in virus replication nor attenuated protein function in vitro, consistent with detection in clinical specimens. A better understanding of the forces governing mumps virus sequence diversity and of the functional consequences of mutations in viral proteins is important for maintaining robust capacity for mumps detection and disease control.


Asunto(s)
Codón de Terminación/genética , Virus de la Parotiditis/fisiología , Mutación , Proteínas Virales/genética , Animales , Humanos , Sarampión/virología , Virulencia , Factores de Virulencia
15.
Open Forum Infect Dis ; 4(4): ofx263, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29308410

RESUMEN

BACKGROUND: Recent mumps outbreaks among 2-dose measles mumps rubella (MMR) vaccine recipients have raised questions regarding the potential benefits of a third dose of vaccine (MMR3). If MMR3 provides a sustained elevation in mumps antibody, it may be beneficial for certain at-risk groups or as an outbreak control measure. METHODS: Sera were collected immediately prior to MMR3 and at 1 month and 1 year post-MMR3 from 656 healthy adults aged 18-28 years in a nonoutbreak setting. Immunoglobulin G (IgG) was measured by enzyme-linked immunosorbent assay (ELISA) using whole mumps virus (commercial ELISA), hemagglutinin (HN; major neutralizing target), and nucleoprotein (NP; immunodominant) antigens. ELISA measurements were compared with in vitro plaque reduction neutralization (PRN) titers, and baseline antibody was compared with post-MMR3 levels. RESULTS: There were modest but statistically significant (P < .05) increases in mumps antibody at 1 month post-MMR3 by all 3 ELISA methods and by PRN titer. At 1 year post-MMR3, mumps antibody declined toward baseline but remained elevated (P < .05). The correlation between PRN titers and ELISA measurements was poor (r2 = .49), although sera with the highest amount of HN IgG also had the highest PRN titers. CONCLUSIONS: Individuals with the lowest baseline PRN titers had the largest increase in frequency of samples that became positive for HN and NP by ELISA. A third dose of MMR may benefit certain individuals with a low level of mumps virus-neutralizing antibody, especially in the context of an outbreak or other high-risk setting. Additionally, poor correlation among serologic tests does not allow effective prediction of PRN titer by ELISA.

16.
J Pediatric Infect Dis Soc ; 4(1): 63-6, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25844166

RESUMEN

Among our cohort of adolescents and young adults with perinatally acquired human immunodeficiency virus, few (17.6%) had measles protective antibodies by plaque reduction neutralization (PRN). Agreement was demonstrated between the commercial enzyme immunoassay and the PRN assay (K = 0.59 [95% confidence interval: 0.23-0.95]). Further studies are needed to understand the determinants of immunity in this population.


Asunto(s)
Susceptibilidad a Enfermedades , Infecciones por VIH/epidemiología , Infecciones por VIH/inmunología , Sarampión/epidemiología , Sarampión/inmunología , Adolescente , Adulto , Antirretrovirales/uso terapéutico , Estudios de Cohortes , Femenino , Infecciones por VIH/tratamiento farmacológico , Humanos , Técnicas para Inmunoenzimas , Masculino , Adulto Joven
17.
Clin Vaccine Immunol ; 21(3): 286-97, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24371258

RESUMEN

Neutralizing antibodies are assumed to be essential for protection against mumps virus infection, but their measurement is labor- and time-intensive. For this reason, enzyme-linked immunosorbent assays (ELISAs) are typically used to measure mumps-specific IgG levels. However, since there is poor correlation between mumps neutralization titers and ELISAs that measure the presence of mumps-specific IgG levels, ELISAs that better correlate with neutralization are needed. To address this issue, we measured mumps antibody levels by plaque reduction neutralization, by a commercial ELISA (whole-virus antigen), and by ELISAs specific for the mumps nucleoprotein and hemagglutinin. The results indicate that differences in the antibody response to the individual mumps proteins could partially explain the lack of correlation among various serologic tests. Furthermore, the data indicate that some seropositive individuals have low levels of neutralizing antibody. If neutralizing antibody is important for protection, this suggests that previous estimates of immunity based on whole-virus ELISAs may be overstated.


Asunto(s)
Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos , Antígenos Virales , Técnicas de Laboratorio Clínico/métodos , Virus de la Parotiditis/inmunología , Paperas/diagnóstico , Paperas/epidemiología , Animales , Anticuerpos Neutralizantes/sangre , Antígenos Virales/inmunología , Preescolar , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Lactante , Masculino , Estudios Seroepidemiológicos
18.
Clin Vaccine Immunol ; 18(1): 35-42, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21047998

RESUMEN

Although high measles, mumps, and rubella (MMR) vaccination coverage has been successful in dramatically reducing mumps disease in the United States, mumps (re)infections occasionally occur in individuals who have been either previously vaccinated or naturally infected. Standard diagnostics that detect virus or virus-specific antibody are dependable for confirming primary mumps infection in immunologically naïve persons, but these methods perform inconsistently for individuals with prior immune exposure. We hypothesized that detection of activated mumps-specific antibody-secreting B cells (ASCs) by enzyme-linked immunospot (ELISPOT) assay could be used as a more reliable diagnostic. To test this, a time course of virus-specific ASC responses was measured by ELISPOT assay following MMR vaccination of 16 previously vaccinated or naturally exposed adult volunteers. Mumps-specific ASCs were detectable in 68% of these individuals at some point during the first 3 weeks following revaccination. In addition, mumps-specific ASCs were detected in 7/7 previously vaccinated individuals who recently had been infected as part of a confirmed mumps outbreak. These data suggest that ELISPOT detection of mumps-specific ASCs has the potential for use as an alternative method of diagnosis when suspect cases cannot be confirmed by detection of IgM or virus. In addition, it was determined that mumps-specific memory B cells are detected at a much lower frequency than measles- or rubella-specific cells, suggesting that mumps infection may not generate robust B-cell memory.


Asunto(s)
Anticuerpos Antivirales/sangre , Linfocitos B/inmunología , Ensayo de Immunospot Ligado a Enzimas/métodos , Vacuna contra el Sarampión-Parotiditis-Rubéola/administración & dosificación , Virus de la Parotiditis/inmunología , Paperas/diagnóstico , Adulto , Anticuerpos Antivirales/inmunología , Células Productoras de Anticuerpos/inmunología , Humanos , Memoria Inmunológica/inmunología , Sarampión/inmunología , Sarampión/prevención & control , Virus del Sarampión/inmunología , Vacuna contra el Sarampión-Parotiditis-Rubéola/inmunología , Persona de Mediana Edad , Paperas/inmunología , Paperas/prevención & control , Rubéola (Sarampión Alemán)/inmunología , Rubéola (Sarampión Alemán)/prevención & control , Virus de la Rubéola/inmunología , Estados Unidos , Vacunación
19.
Virology ; 363(2): 319-32, 2007 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-17336362

RESUMEN

Treatment of wild type vaccinia virus infected cells with the anti-poxviral drug isatin-beta-thiosemicarbazone (IBT) induces the viral postreplicative transcription apparatus to synthesize longer-than-normal mRNAs through an unknown mechanism. Previous studies have shown that virus mutants resistant to or dependent on IBT affect genes involved in control of viral postreplicative transcription elongation. This study was initiated in order to identify additional viral genes involved in control of vaccinia postreplicative transcription elongation. Eight independent, spontaneous IBT resistant mutants of vaccinia virus were isolated. Marker rescue experiments mapped two mutants to gene G2R, which encodes a previously characterized postreplicative gene positive transcription elongation factor. Three mutants mapped to the largest subunit of the viral RNA polymerase, rpo147, the product of gene J6R. One mutant contained missense mutations in both G2R and A24R (rpo132, the second largest subunit of the RNA polymerase). Two mutants could not be mapped, however sequence analysis demonstrated that neither of these mutants contained mutations in previously identified IBT resistance or dependence genes. Phenotypic and biochemical analysis of the mutants suggests that they possess defects in transcription elongation that compensate for the elongation enhancing effects of IBT. The results implicate the largest subunit of the RNA polymerase (rpo147) in the control of elongation, and suggest that there exist additional gene products which mediate intermediate and late transcription elongation in vaccinia virus.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Regulación Viral de la Expresión Génica , Virus Vaccinia/genética , Proteínas Virales/genética , Animales , Dominio Catalítico/genética , Línea Celular , ARN Polimerasas Dirigidas por ADN/química , Farmacorresistencia Viral , Perfilación de la Expresión Génica , Humanos , Isatina/análogos & derivados , Isatina/farmacología , Modelos Químicos , Mutación , Factores de Elongación de Péptidos , Fenotipo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Factores de Elongación Transcripcional/genética , Virus Vaccinia/efectos de los fármacos , Proteínas Virales/química
20.
J Virol ; 78(20): 10953-9, 2004 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15452215

RESUMEN

Unlike naive CD8+ T cells, antigen-experienced memory CD8+ T cells persist over time due to their unique ability to homeostatically proliferate. It was hypothesized that memory cells might differentially regulate the expression of genes that control the cell cycle to facilitate homeostatic proliferation. To test this, the expression levels of 96 different cell cycle regulatory genes were compared between transgenic naive and memory CD8+ T cells that specifically recognize the GP33-41 epitope of lymphocytic choriomeningitis virus (LCMV). It was discovered that relative to naive cells, memory cells overexpress several important genes that control the transition between G(1) and S phase. Some of these genes include those encoding cyclins D3, D2, B1, C, and H, cyclin-dependent kinases (cdk's) 4 and 6, the cdk inhibitors p16, p15, and p18, and other genes involved in protein degradation and DNA replication. Importantly, these differences were observed both in total populations of LCMV-specific naive and memory CD8+ cells and in LCMV-specific CD8+ T-cell populations that were in the G(1) phase of the cell cycle only. In addition, the expression differences between naive and memory cells were exaggerated following antigenic stimulation. The fact that memory cells are precharged with several of the major factors that are necessary for the G(1)- to-S-phase transition suggests they may require a lower threshold of stimulation to enter the cell cycle.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Regulación de la Expresión Génica , Genes cdc/fisiología , Memoria Inmunológica , Virus de la Coriomeningitis Linfocítica/inmunología , Proteínas/metabolismo , Animales , Linfocitos T CD8-positivos/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Fase G1 , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Proteínas/genética
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