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1.
Nature ; 592(7852): 122-127, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33636719

RESUMEN

During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.


Asunto(s)
COVID-19/transmisión , COVID-19/virología , Mutación , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/genética , Replicación Viral/genética , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Bronquios/citología , Bronquios/virología , COVID-19/epidemiología , Línea Celular , Células Cultivadas , Cricetinae , Modelos Animales de Enfermedad , Células Epiteliales/virología , Femenino , Hurones/virología , Efecto Fundador , Técnicas de Sustitución del Gen , Aptitud Genética , Humanos , Masculino , Mesocricetus , Ratones , Mucosa Nasal/citología , Mucosa Nasal/virología , Unión Proteica , ARN Viral/análisis , Receptores de Coronavirus/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad
2.
PLoS Pathog ; 20(4): e1012131, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38626244

RESUMEN

Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype swine IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intrahost Single Nucleotide Variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics.


Asunto(s)
Flujo Genético , Infecciones por Orthomyxoviridae , Enfermedades de los Porcinos , Animales , Porcinos , Infecciones por Orthomyxoviridae/virología , Enfermedades de los Porcinos/virología , Subtipo H3N2 del Virus de la Influenza A/genética , Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Variación Genética , Evolución Molecular , Selección Genética , Filogenia
3.
MMWR Morb Mortal Wkly Rep ; 71(10): 365-370, 2022 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-35271561

RESUMEN

In the United States, annual vaccination against seasonal influenza is recommended for all persons aged ≥6 months except when contraindicated (1). Currently available influenza vaccines are designed to protect against four influenza viruses: A(H1N1)pdm09 (the 2009 pandemic virus), A(H3N2), B/Victoria lineage, and B/Yamagata lineage. Most influenza viruses detected this season have been A(H3N2) (2). With the exception of the 2020-21 season, when data were insufficient to generate an estimate, CDC has estimated the effectiveness of seasonal influenza vaccine at preventing laboratory-confirmed, mild/moderate (outpatient) medically attended acute respiratory infection (ARI) each season since 2004-05. This interim report uses data from 3,636 children and adults with ARI enrolled in the U.S. Influenza Vaccine Effectiveness Network during October 4, 2021-February 12, 2022. Overall, vaccine effectiveness (VE) against medically attended outpatient ARI associated with influenza A(H3N2) virus was 16% (95% CI = -16% to 39%), which is considered not statistically significant. This analysis indicates that influenza vaccination did not reduce the risk for outpatient medically attended illness with influenza A(H3N2) viruses that predominated so far this season. Enrollment was insufficient to generate reliable VE estimates by age group or by type of influenza vaccine product (1). CDC recommends influenza antiviral medications as an adjunct to vaccination; the potential public health benefit of antiviral medications is magnified in the context of reduced influenza VE. CDC routinely recommends that health care providers continue to administer influenza vaccine to persons aged ≥6 months as long as influenza viruses are circulating, even when VE against one virus is reduced, because vaccine can prevent serious outcomes (e.g., hospitalization, intensive care unit (ICU) admission, or death) that are associated with influenza A(H3N2) virus infection and might protect against other influenza viruses that could circulate later in the season.


Asunto(s)
Subtipo H3N2 del Virus de la Influenza A/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/prevención & control , Eficacia de las Vacunas , Adolescente , Adulto , Anciano , Niño , Preescolar , Humanos , Lactante , Subtipo H1N1 del Virus de la Influenza A/inmunología , Virus de la Influenza B/inmunología , Persona de Mediana Edad , Vigilancia de la Población , Estaciones del Año , Estados Unidos/epidemiología , Vacunación
4.
MMWR Morb Mortal Wkly Rep ; 71(6): 206-211, 2022 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-35143464

RESUMEN

Genomic surveillance is a critical tool for tracking emerging variants of SARS-CoV-2 (the virus that causes COVID-19), which can exhibit characteristics that potentially affect public health and clinical interventions, including increased transmissibility, illness severity, and capacity for immune escape. During June 2021-January 2022, CDC expanded genomic surveillance data sources to incorporate sequence data from public repositories to produce weighted estimates of variant proportions at the jurisdiction level and refined analytic methods to enhance the timeliness and accuracy of national and regional variant proportion estimates. These changes also allowed for more comprehensive variant proportion estimation at the jurisdictional level (i.e., U.S. state, district, territory, and freely associated state). The data in this report are a summary of findings of recent proportions of circulating variants that are updated weekly on CDC's COVID Data Tracker website to enable timely public health action.† The SARS-CoV-2 Delta (B.1.617.2 and AY sublineages) variant rose from 1% to >50% of viral lineages circulating nationally during 8 weeks, from May 1-June 26, 2021. Delta-associated infections remained predominant until being rapidly overtaken by infections associated with the Omicron (B.1.1.529 and BA sublineages) variant in December 2021, when Omicron increased from 1% to >50% of circulating viral lineages during a 2-week period. As of the week ending January 22, 2022, Omicron was estimated to account for 99.2% (95% CI = 99.0%-99.5%) of SARS-CoV-2 infections nationwide, and Delta for 0.7% (95% CI = 0.5%-1.0%). The dynamic landscape of SARS-CoV-2 variants in 2021, including Delta- and Omicron-driven resurgences of SARS-CoV-2 transmission across the United States, underscores the importance of robust genomic surveillance efforts to inform public health planning and practice.


Asunto(s)
COVID-19/epidemiología , COVID-19/virología , SARS-CoV-2/genética , Centers for Disease Control and Prevention, U.S. , Genómica , Humanos , Prevalencia , Vigilancia en Salud Pública/métodos , Estados Unidos/epidemiología
5.
Clin Infect Dis ; 73(11): e4244-e4250, 2021 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-33367650

RESUMEN

BACKGROUND: At the start of the 2019-2020 influenza season, concern arose that circulating B/Victoria viruses of the globally emerging clade V1A.3 were antigenically drifted from the strain included in the vaccine. Intense B/Victoria activity was followed by circulation of genetically diverse A(H1N1)pdm09 viruses that were also antigenically drifted. We measured vaccine effectiveness (VE) in the United States against illness from these emerging viruses. METHODS: We enrolled outpatients aged ≥6 months with acute respiratory illness at 5 sites. Respiratory specimens were tested for influenza by reverse-transcriptase polymerase chain reaction (RT-PCR). Using the test-negative design, we determined influenza VE by virus subtype/lineage and genetic subclades by comparing odds of vaccination in influenza cases versus test-negative controls. RESULTS: Among 8845 enrollees, 2722 (31%) tested positive for influenza, including 1209 (44%) for B/Victoria and 1405 (51%) for A(H1N1)pdm09. Effectiveness against any influenza illness was 39% (95% confidence interval [CI]: 32-44), 45% (95% CI: 37-52) against B/Victoria and 30% (95% CI: 21-39) against A(H1N1)pdm09-associated illness. Vaccination offered no protection against A(H1N1)pdm09 viruses with antigenically drifted clade 6B.1A 183P-5A+156K HA genes (VE 7%; 95% CI: -14 to 23%) which predominated after January. CONCLUSIONS: Vaccination provided protection against influenza illness, mainly due to infections from B/Victoria viruses. Vaccine protection against illness from A(H1N1)pdm09 was lower than historically observed effectiveness of 40%-60%, due to late-season vaccine mismatch following emergence of antigenically drifted viruses. The effect of drift on vaccine protection is not easy to predict and, even in drifted years, significant protection can be observed.


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Deriva y Cambio Antigénico , Humanos , Lactante , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza B , Gripe Humana/epidemiología , Gripe Humana/prevención & control , Estaciones del Año , Estados Unidos/epidemiología , Vacunación , Eficacia de las Vacunas
6.
J Infect Dis ; 221(1): 8-15, 2020 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-31665373

RESUMEN

BACKGROUND: Increased illness due to antigenically drifted A(H3N2) clade 3C.3a influenza viruses prompted concerns about vaccine effectiveness (VE) and vaccine strain selection. We used US virologic surveillance and US Influenza Vaccine Effectiveness (Flu VE) Network data to evaluate consequences of this clade. METHODS: Distribution of influenza viruses was described using virologic surveillance data. The Flu VE Network enrolled ambulatory care patients aged ≥6 months with acute respiratory illness at 5 sites. Respiratory specimens were tested for influenza by means of reverse-transcriptase polymerase chain reaction and were sequenced. Using a test-negative design, we estimated VE, comparing the odds of influenza among vaccinated versus unvaccinated participants. RESULTS: During the 2018-2019 influenza season, A(H3N2) clade 3C.3a viruses caused an increasing proportion of influenza cases. Among 2763 Flu VE Network case patients, 1325 (48%) were infected with A(H1N1)pdm09 and 1350 (49%) with A(H3N2); clade 3C.3a accounted for 977 (93%) of 1054 sequenced A(H3N2) viruses. VE was 44% (95% confidence interval, 37%-51%) against A(H1N1)pdm09 and 9% (-4% to 20%) against A(H3N2); VE was 5% (-10% to 19%) against A(H3N2) clade 3C.3a viruses. CONCLUSIONS: The predominance of A(H3N2) clade 3C.3a viruses during the latter part of the 2018-2019 season was associated with decreased VE, supporting the A(H3N2) vaccine component update for 2019-2020 northern hemisphere influenza vaccines.


Asunto(s)
Variación Antigénica , Subtipo H1N1 del Virus de la Influenza A , Subtipo H3N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza , Gripe Humana/epidemiología , Gripe Humana/prevención & control , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Humanos , Lactante , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Gripe Humana/virología , Masculino , Persona de Mediana Edad , Nariz/virología , Orofaringe/virología , Vigilancia de la Población , ARN Viral/análisis , Estados Unidos/epidemiología , Vacunación , Adulto Joven
7.
J Virol ; 93(1)2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30305359

RESUMEN

The fifth wave of the H7N9 influenza epidemic in China was distinguished by a sudden increase in human infections, an extended geographic distribution, and the emergence of highly pathogenic avian influenza (HPAI) viruses. Genetically, some H7N9 viruses from the fifth wave have acquired novel amino acid changes at positions involved in mammalian adaptation, antigenicity, and hemagglutinin cleavability. Here, several human low-pathogenic avian influenza (LPAI) and HPAI H7N9 virus isolates from the fifth epidemic wave were assessed for their pathogenicity and transmissibility in mammalian models, as well as their ability to replicate in human airway epithelial cells. We found that an LPAI virus exhibited a similar capacity to replicate and cause disease in two animal species as viruses from previous waves. In contrast, HPAI H7N9 viruses possessed enhanced virulence, causing greater lethargy and mortality, with an extended tropism for brain tissues in both ferret and mouse models. These HPAI viruses also showed signs of adaptation to mammalian hosts by acquiring the ability to fuse at a lower pH threshold than other H7N9 viruses. All of the fifth-wave H7N9 viruses were able to transmit among cohoused ferrets but exhibited a limited capacity to transmit by respiratory droplets, and deep sequencing analysis revealed that the H7N9 viruses sampled after transmission showed a reduced amount of minor variants. Taken together, we conclude that the fifth-wave HPAI H7N9 viruses have gained the ability to cause enhanced disease in mammalian models and with further adaptation may acquire the ability to cause an H7N9 pandemic.IMPORTANCE The potential pandemic risk posed by avian influenza H7N9 viruses was heightened during the fifth epidemic wave in China due to the sudden increase in the number of human infections and the emergence of antigenically distinct LPAI and HPAI H7N9 viruses. In this study, a group of fifth-wave HPAI and LPAI viruses was evaluated for its ability to infect, cause disease, and transmit in small-animal models. The ability of HPAI H7N9 viruses to cause more severe disease and to replicate in brain tissues in animal models as well as their ability to fuse at a lower pH threshold than LPAI H7N9 viruses suggests that the fifth-wave H7N9 viruses have evolved to acquire novel traits with the potential to pose a higher risk to humans. Although the fifth-wave H7N9 viruses have not yet gained the ability to transmit efficiently by air, continuous surveillance and risk assessment remain essential parts of our pandemic preparedness efforts.


Asunto(s)
Subtipo H7N9 del Virus de la Influenza A/patogenicidad , Gripe Humana/virología , Infecciones por Orthomyxoviridae/epidemiología , ARN Viral/genética , Análisis de Secuencia de ARN/métodos , Animales , Línea Celular , China/epidemiología , Chlorocebus aethiops , Epidemias , Evolución Molecular , Hurones , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Subtipo H7N9 del Virus de la Influenza A/genética , Gripe Humana/epidemiología , Gripe Humana/transmisión , Ratones , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/virología , Medición de Riesgo , Células Vero , Tropismo Viral , Virulencia
8.
Mol Cell ; 48(2): 195-206, 2012 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-22959275

RESUMEN

LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.


Asunto(s)
Empalme Alternativo/genética , ARN Mensajero , Proteínas de Unión al ARN , Sitios de Unión/genética , Células Madre Embrionarias , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Motivos de Nucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo
9.
J Virol ; 92(11)2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-29540597

RESUMEN

Influenza A(H1) viruses circulating in swine represent an emerging virus threat, as zoonotic infections occur sporadically following exposure to swine. A fatal infection caused by an H1N1 variant (H1N1v) virus was detected in a patient with reported exposure to swine and who presented with pneumonia, respiratory failure, and cardiac arrest. To understand the genetic and phenotypic characteristics of the virus, genome sequence analysis, antigenic characterization, and ferret pathogenesis and transmissibility experiments were performed. Antigenic analysis of the virus isolated from the fatal case, A/Ohio/09/2015, demonstrated significant antigenic drift away from the classical swine H1N1 variant viruses and H1N1 pandemic 2009 viruses. A substitution in the H1 hemagglutinin (G155E) was identified that likely impacted antigenicity, and reverse genetics was employed to understand the molecular mechanism of antibody escape. Reversion of the substitution to 155G, in a reverse genetics A/Ohio/09/2015 virus, showed that this residue was central to the loss of hemagglutination inhibition by ferret antisera raised against a prototypical H1N1 pandemic 2009 virus (A/California/07/2009), as well as gamma lineage classical swine H1N1 viruses, demonstrating the importance of this residue for antibody recognition of this H1 lineage. When analyzed in the ferret model, A/Ohio/09/2015 and another H1N1v virus, A/Iowa/39/2015, as well as A/California/07/2009, replicated efficiently in the respiratory tract of ferrets. The two H1N1v viruses transmitted efficiently among cohoused ferrets, but respiratory droplet transmission studies showed that A/California/07/2009 transmitted through the air more efficiently. Preexisting immunity to A/California/07/2009 did not fully protect ferrets from challenge with A/Ohio/09/2015.IMPORTANCE Human infections with classical swine influenza A(H1N1) viruses that circulate in pigs continue to occur in the United States following exposure to swine. To understand the genetic and virologic characteristics of a virus (A/Ohio/09/2015) associated with a fatal infection and a virus associated with a nonfatal infection (A/Iowa/39/2015), we performed genome sequence analysis, antigenic testing, and pathogenicity and transmission studies in a ferret model. Reverse genetics was employed to identify a single antigenic site substitution (HA G155E) responsible for antigenic variation of A/Ohio/09/2015 compared to related classical swine influenza A(H1N1) viruses. Ferrets with preexisting immunity to the pandemic A(H1N1) virus were challenged with A/Ohio/09/2015, demonstrating decreased protection. These data illustrate the potential for currently circulating swine influenza viruses to infect and cause illness in humans with preexisting immunity to H1N1 pandemic 2009 viruses and a need for ongoing risk assessment and development of candidate vaccine viruses for improved pandemic preparedness.


Asunto(s)
Variación Antigénica/genética , Hurones/virología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/veterinaria , Animales , Variación Antigénica/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/clasificación , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Porcinos/virología , Enfermedades de los Porcinos/virología
10.
Nano Lett ; 18(5): 2802-2806, 2018 05 09.
Artículo en Inglés | MEDLINE | ID: mdl-29683680

RESUMEN

The fabrication of metallic electromagnetic meta-atoms on a soft microstructured polymer scaffold using a MEMS-based stencil lithography technique is demonstrated. Using this technique, complex metasurfaces that are generally impossible to fabricate with traditional photolithographic techniques are created. By engineering the mechanical deformation of the polymer scaffold, the metasurface reflectivity in the mid-infrared can be tuned by the application of moderate strains.

11.
J Clin Microbiol ; 55(12): 3492-3501, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-28978683

RESUMEN

Influenza A and B viruses are the causative agents of annual influenza epidemics that can be severe, and influenza A viruses intermittently cause pandemics. Sequence information from influenza virus genomes is instrumental in determining mechanisms underpinning antigenic evolution and antiviral resistance. However, due to sequence diversity and the dynamics of influenza virus evolution, rapid and high-throughput sequencing of influenza viruses remains a challenge. We developed a single-reaction influenza A/B virus (FluA/B) multiplex reverse transcription-PCR (RT-PCR) method that amplifies the most critical genomic segments (hemagglutinin [HA], neuraminidase [NA], and matrix [M]) of seasonal influenza A and B viruses for next-generation sequencing, regardless of viral type, subtype, or lineage. Herein, we demonstrate that the strategy is highly sensitive and robust. The strategy was validated on thousands of seasonal influenza A and B virus-positive specimens using multiple next-generation sequencing platforms.


Asunto(s)
Virus de la Influenza A/clasificación , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza B/clasificación , Virus de la Influenza B/aislamiento & purificación , Gripe Humana/virología , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Monitoreo Epidemiológico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Virus de la Influenza A/genética , Virus de la Influenza B/genética , Epidemiología Molecular/métodos
12.
J Virol ; 88(8): 4021-39, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24453373

RESUMEN

UNLABELLED: Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments. It is likely that the stage of fetal development, as well as the state of differentiation of susceptible cells at the time of infection, affects the severity of the disease. We used human embryonic stem (ES) cell-derived primitive prerosette neural stem cells (pNSCs) and neural progenitor cells (NPCs) maintained in chemically defined conditions to study HCMV replication in cells at the early stages of neural development. In contrast to what was observed previously using fetus-derived NPCs, infection of ES cell-derived pNSCs with HCMV was nonprogressive. At a low multiplicity of infection, we observed only a small percentage of cells expressing immediate-early genes (IE) and early genes. IE expression was found to be restricted to cells negative for the anterior marker FORSE-1, and treatment of pNSCs with retinoic acid restored IE expression. Differentiation of pNSCs into NPCs restored IE expression but not the transactivation of early genes. Virions produced in NPCs and pNSCs were exclusively cell associated and were mostly non-neural tropic. Finally, we found that viral genomes could persist in pNSC cultures for up to a month after infection despite the absence of detectable IE expression by immunofluorescence, and infectious virus could be produced upon differentiation of pNSCs to neurons. In conclusion, our results highlight the complex array of hurdles that HCMV must overcome in order to infect primitive neural stem cells and suggest that these cells might act as a reservoir for the virus. IMPORTANCE: Human cytomegalovirus (HCMV) is a betaherpesvirus that is highly prevalent in the population. HCMV infection is usually asymptomatic but can lead to severe consequences in immunosuppressed individuals. HCMV is also the most important infectious cause of congenital developmental birth defects. Manifestations of fetal HCMV disease range from deafness and learning disabilities to more severe symptoms such as microcephaly. In this study, we have used embryonic stem cells to generate primitive neural stem cells and have used these to model HCMV infection of the fetal central nervous system (CNS) in vitro. Our results reveal that these cells, which are similar to those present in the developing neural tube, do not support viral replication but instead likely constitute a viral reservoir. Future work will define the effect of viral persistence on cellular functions as well as the exogenous signals leading to the reactivation of viral replication in the CNS.


Asunto(s)
Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , ADN Viral/metabolismo , Células Madre Embrionarias/virología , Células-Madre Neurales/virología , Diferenciación Celular , Citomegalovirus/genética , Infecciones por Citomegalovirus/fisiopatología , ADN Viral/genética , Células Madre Embrionarias/citología , Femenino , Humanos , Células-Madre Neurales/citología , Replicación Viral
14.
J Virol ; 86(1): 226-35, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22013051

RESUMEN

Human cytomegalovirus (HCMV) contributes its own set of microRNAs (miRNAs) during lytic infection of cells, likely fine-tuning conditions important for viral replication. To enhance our understanding of this component of the HCMV-host transcriptome, we have conducted deep-sequencing analysis of small RNAs (smRNA-seq) from infected human fibroblast cells. We found that HCMV-encoded miRNAs accumulate to ∼20% of the total smRNA population at late stages of infection, and our analysis led to improvements in viral miRNA annotations and identification of two novel HCMV miRNAs, miR-US22 and miR-US33as. Both of these miRNAs were capable of functionally repressing synthetic targets in transient transfection experiments. Additionally, through cross-linking and immunoprecipitation (CLIP) of Argonaute (Ago)-bound RNAs from infected cells, followed by high-throughput sequencing, we have obtained direct evidence for incorporation of all HCMV miRNAs into the endogenous host silencing machinery. Surprisingly, three HCMV miRNA precursors exhibited differential incorporation of their mature miRNA arms between Ago2 and Ago1 complexes. Host miRNA abundances were also affected by HCMV infection, with significant upregulation observed for an miRNA cluster containing miR-96, miR-182, and miR-183. In addition to miRNAs, we also identified novel forms of virus-derived smRNAs, revealing greater complexity within the smRNA population during HCMV infection.


Asunto(s)
Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , Citomegalovirus/genética , Perfilación de la Expresión Génica , MicroARNs/genética , ARN Viral/genética , Secuencia de Bases , Línea Celular , Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/metabolismo , Interacciones Huésped-Patógeno , Humanos , MicroARNs/metabolismo , Datos de Secuencia Molecular , ARN Viral/metabolismo , Análisis de Secuencia de ARN
15.
Microbiol Spectr ; 11(4): e0116623, 2023 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-37404140

RESUMEN

While primarily considered a respiratory pathogen, influenza A virus (IAV) is nonetheless capable of spreading to, and replicating in, numerous extrapulmonary tissues in humans. However, within-host assessments of genetic diversity during multicycle replication have been largely limited to respiratory tract tissues and specimens. As selective pressures can vary greatly between anatomical sites, there is a need to examine how measures of viral diversity may vary between influenza viruses exhibiting different tropisms in humans, as well as following influenza virus infection of cells derived from different organ systems. Here, we employed human primary tissue constructs emulative of the human airway or corneal surface, and we infected both with a panel of human- and avian-origin IAV, inclusive of H1 and H3 subtype human viruses and highly pathogenic H5 and H7 subtype viruses, which are associated with both respiratory disease and conjunctivitis following human infection. While both cell types supported productive replication of all viruses, airway-derived tissue constructs elicited greater induction of genes associated with antiviral responses than did corneal-derived constructs. We used next-generation sequencing to examine viral mutations and population diversity, utilizing several metrics. With few exceptions, generally comparable measures of viral diversity and mutational frequency were detected following homologous virus infection of both respiratory-origin and ocular-origin tissue constructs. Expansion of within-host assessments of genetic diversity to include IAV with atypical clinical presentations in humans or in extrapulmonary cell types can provide greater insight into understanding those features most prone to modulation in the context of viral tropism. IMPORTANCE Influenza A virus (IAV) can infect tissues both within and beyond the respiratory tract, leading to extrapulmonary complications, such as conjunctivitis or gastrointestinal disease. Selective pressures governing virus replication and induction of host responses can vary based on the anatomical site of infection, yet studies examining within-host assessments of genetic diversity are typically only conducted in cells derived from the respiratory tract. We examined the contribution of influenza virus tropism on these properties two different ways: by using IAV associated with different tropisms in humans, and by infecting human cell types from two different organ systems susceptible to IAV infection. Despite the diversity of cell types and viruses employed, we observed generally similar measures of viral diversity postinfection across all conditions tested; these findings nonetheless contribute to a greater understanding of the role tissue type contributes to the dynamics of virus evolution within a human host.


Asunto(s)
Conjuntivitis , Virus de la Influenza A , Gripe Humana , Animales , Humanos , Perros , Virus de la Influenza A/genética , Sistema Respiratorio , Células de Riñón Canino Madin Darby
16.
bioRxiv ; 2023 Oct 25.
Artículo en Inglés | MEDLINE | ID: mdl-37961583

RESUMEN

Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intra-host single nucleotide variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics.

17.
bioRxiv ; 2020 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-33140052

RESUMEN

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic 1 . However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro , it provides a real competitive advantage in vivo , particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

18.
J Virol Methods ; 270: 95-105, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31004662

RESUMEN

Influenza A virus is a negative-sense RNA virus with a segmented genome consisting of eight RNA segments. Avian influenza A virus (AIV) primarily infects avian hosts and sporadically infects mammals, which can lead to adaptation to new species. Next-generation sequencing (NGS) of emerging AIV genomes extracted from respiratory samples collected on sequential days from animal models and clinical patients enables analysis of the emergence of evolutionary variants within the virus population over time. However, obtaining codon complete AIV genome at a sufficient coverage depth for nucleotide variant calling remains a challenge, especially from post-inoculation respiratory samples collected at late time points that have low viral titers. In this study, nasal wash samples from ferrets inoculated with different subtypes of AIV were collected on various days post-inoculation. Each nasal wash sample was aliquoted and extracted using five commercially available nucleic acid extraction methods. Extracted influenza virus RNA was amplified and NGS conducted using Illumina Mi-Seq. For each nasal wash sample, completeness of AIV genome segments and coverage depth were compared among five extraction methods. Nucleic acids extracted by MagNA pure compact RNA isolation consistently yielded codon complete sequences for all eight genome segments at the required coverage depth at each time point sampled. The study revealed that DNase treatment was critical to the amplification of influenza genome segments and the downstream success of codon complete NGS from nasal wash samples. The findings from this study can be applied to improve NGS of influenza and other RNA viruses that infect the respiratory tract and are collected from respiratory samples.


Asunto(s)
Hurones/virología , Secuenciación de Nucleótidos de Alto Rendimiento , Virus de la Influenza A/aislamiento & purificación , Ácidos Nucleicos/aislamiento & purificación , Extracción en Fase Sólida/métodos , Animales , Genoma Viral , Virus de la Influenza A/genética , ARN Viral/aislamiento & purificación
19.
Virology ; 534: 36-44, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31176062

RESUMEN

Bangladesh has reported repeated outbreaks of highly pathogenic avian influenza (HPAI) A(H5) viruses in poultry since 2007. Because of the large number of live poultry markets (LPM) relative to the population density of poultry throughout the country, these markets can serve as sentinel sites for HPAI A(H5) detection. Through active LPM surveillance during June 2016-June 2017, HPAI A(H5N6) viruses along with 14 other subtypes of influenza A viruses were detected. The HPAI A(H5N6) viruses belonged to clade 2.3.4.4 and were likely introduced into Bangladesh around March 2016. Human infections with influenza clade 2.3.4.4 viruses in Bangladesh have not been identified, but the viruses had several molecular markers associated with potential human infection. Vigilant surveillance at the animal-human interface is essential to identify emerging avian influenza viruses with the potential to threaten public and animal health.


Asunto(s)
Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Enfermedades de las Aves de Corral/virología , Animales , Bangladesh/epidemiología , Brotes de Enfermedades , Patos , Evolución Molecular , Gansos/virología , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Gripe Aviar/epidemiología , Filogenia , Enfermedades de las Aves de Corral/epidemiología
20.
Sci Rep ; 8(1): 15746, 2018 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-30341398

RESUMEN

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

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