RESUMEN
Some respiratory viruses can cause a viral interference through the activation of the interferon (IFN) pathway that reduces the replication of another virus. Epidemiological studies of coinfections between SARS-CoV-2 and other respiratory viruses have been hampered by non-pharmacological measures applied to mitigate the spread of SARS-CoV-2 during the COVID-19 pandemic. With the ease of these interventions, SARS-CoV-2 and influenza A viruses can now co-circulate. It is thus of prime importance to characterize their interactions. In this work, we investigated viral interference effects between an Omicron variant and a contemporary influenza A/H3N2 strain, in comparison with an ancestral SARS-CoV-2 strain and the 2009 pandemic influenza A/H1N1 virus. We infected nasal human airway epitheliums with SARS-CoV-2 and influenza, either simultaneously or 24 h apart. Viral load was measured by RT-qPCR and IFN-α/ß/λ1/λ2 proteins were quantified by immunoassay. Expression of four interferon-stimulated genes (ISGs; OAS1/IFITM3/ISG15/MxA) was also measured by RT-droplet digital PCR. Additionally, susceptibility of each virus to IFN-α/ß/λ2 recombinant proteins was determined. Our results showed that influenza A, and especially A/H3N2, interfered with both SARS-CoV-2 viruses, but that SARS-CoV-2 did not significantly interfere with A/H3N2 or A/H1N1. Consistently with these results, influenza, and particularly the A/H3N2 strain, caused a higher production of IFN proteins and expression of ISGs than SARS-CoV-2. SARS-CoV-2 induced a marginal IFN production and reduced the IFN response during coinfections with influenza. All viruses were susceptible to exogenous IFNs, with the ancestral SARS-CoV-2 and Omicron being less susceptible to type I and type III IFNs, respectively. Thus, influenza A causes a viral interference towards SARS-CoV-2 most likely through an IFN response. The opposite is not necessarily true, and a concurrent infection with both viruses leads to a lower IFN response. Taken together, these results help us to understand how SARS-CoV-2 interacts with another major respiratory pathogen.
Asunto(s)
COVID-19 , Coinfección , Subtipo H1N1 del Virus de la Influenza A , Subtipo H3N2 del Virus de la Influenza A , Gripe Humana , SARS-CoV-2 , Interferencia Viral , Humanos , COVID-19/virología , Gripe Humana/virología , Subtipo H3N2 del Virus de la Influenza A/genética , Coinfección/virología , Subtipo H1N1 del Virus de la Influenza A/genética , Interferones/metabolismo , Carga Viral , Replicación Viral , Virus de la Influenza ARESUMEN
OBJECTIVES: COVID-19 has been associated with preterm birth (PTB) and placental-mediated complications, including fetal growth restriction and preeclampsia (PE). This study aimed to estimate the impact of COVID-19 and vaccination on adverse pregnancy outcomes and markers of placental function. METHODS: We performed a study on a prospective cohort of women recruited in the first trimester of pregnancy during the early COVID-19 pandemic period (December 2020 to December 2021). At each trimester of pregnancy, the assessment included a questionnaire on COVID-19 and vaccination status; serological tests for COVID-19 (for asymptomatic infection); measurement of placental growth factor (PlGF) and soluble fms-like tyrosine kinase 1 (sFlt-1) in maternal blood; measurement of mean uterine artery pulsatility index (UtA-PI); and pregnancy outcomes (PTB, PE, birth weight below the fifth and the tenth percentile). RESULTS: Among 788 patients with complete data, we observed 101 (13%) cases of symptomatic infection and 74 (9%) cases of asymptomatic infection with SARS-CoV-2. Most cases (73%) of infection were among women with previous vaccination or COVID-19 infection before pregnancy. COVID-19 infection was not associated with adverse pregnancy outcomes, abnormal fetal growth, sFlt-1/PlGF ratio, or mean UtA-PI. Vaccination during pregnancy did not influence these outcomes either. We observed no case of severe COVID-19 infection requiring respiratory support. CONCLUSION: Mild symptomatic or asymptomatic COVID-19 during pregnancy did not influence the risk of adverse pregnancy outcomes and the markers of placental function in predominantly vaccinated women. Fetal growth monitoring is unlikely to be mandatory in women with mild symptoms of COVID-19.
RESUMEN
In vitro selection of remdesivir-resistant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed the emergence of a V166L substitution, located outside of the polymerase active site of the Nsp12 protein, after 9 passages of a single lineage. V166L remained the only Nsp12 substitution after 17 passages (10 µM remdesivir), conferring a 2.3-fold increase in 50% effective concentration (EC50). When V166L was introduced into a recombinant SARS-CoV-2 virus, a 1.5-fold increase in EC50 was observed, indicating a high in vitro barrier to remdesivir resistance.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/química , Alanina/análogos & derivados , Alanina/metabolismo , Antivirales/química , HumanosRESUMEN
Human respiratory syncytial virus (HRSV) constitutes one the main causes of respiratory infection in neonates and infants worldwide. Transcriptome analysis of clinical samples using high-throughput technologies remains an important tool to better understand virus-host complex interactions in the real-life setting but also to identify new diagnosis/prognosis markers or therapeutics targets. A major challenge when exploiting clinical samples such as nasal swabs, washes, or bronchoalveolar lavages is the poor quantity and integrity of nucleic acids. In this study, we applied a tailored transcriptomics workflow to exploit nasal wash samples from children who tested positive for HRSV. Our analysis revealed a characteristic immune signature as a direct reflection of HRSV pathogenesis and highlighted putative biomarkers of interest such as IP-10, TMEM190, MCEMP1, and TIMM23.
Asunto(s)
Infecciones por Virus Sincitial Respiratorio , Infecciones del Sistema Respiratorio , Niño , Perfilación de la Expresión Génica , Humanos , Lactante , Recién Nacido , Nasofaringe , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/inmunologíaRESUMEN
The polymerase acidic (PA) I38T substitution is a dominant marker of resistance to baloxavir. We evaluated the impact of I38T on the fitness of a contemporary influenza A(H3N2) virus. Influenza A/Switzerland/9715293/2013 (H3N2) wild-type (WT) virus and its I38T mutant were rescued by reverse genetics. Replication kinetics were compared using ST6GalI-MDCK and A549 cells and infectivity/contact transmissibility were evaluated in guinea pigs. Nasal wash (NW) viral titres were determined by TCID50 ml-1 in ST6GalI-MDCK cells. Competition experiments were performed and the evolution of viral population was assessed by droplet digital RT-PCR. I38T did not alter in vitro replication. I38T induced comparable titres vs the WT in guinea pigs NWs and the two viruses transmitted equally by direct contact. However, a 50â%:50â% mixture inoculum evolved to mean WT/I38T ratios of 71â%:29â% and 66.4â%:33.6â% on days 4 and 6 p.i., respectively. Contemporary influenza A(H3N2)-I38T PA variants may conserve a significant level of viral fitness.
Asunto(s)
Subtipo H3N2 del Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/virología , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genética , Células A549 , Sustitución de Aminoácidos , Animales , Antivirales/farmacología , Dibenzotiepinas/farmacología , Perros , Farmacorresistencia Viral , Cobayas , Humanos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Células de Riñón Canino Madin Darby , Morfolinas/farmacología , Nariz/virología , Infecciones por Orthomyxoviridae/transmisión , Piridonas/farmacología , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Genética Inversa , Triazinas/farmacología , Carga Viral , Proteínas Virales/química , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
BACKGROUND: Zika virus (ZIKV) has been associated with several neurological complications in adult patients. METHODS: We used a mouse model deficient in TRIF and IPS-1 adaptor proteins, which are involved in type I interferon production, to study the role of microglia during brain infection by ZIKV. Young adult mice were infected intravenously with the contemporary ZIKV strain PRVABC59 (1 × 105 PFUs/100 µL). RESULTS: Infected mice did not present overt clinical signs of the disease nor body weight loss compared with noninfected animals. However, mice exhibited a viremia and a brain viral load that were maximal (1.3 × 105 genome copies/mL and 9.8 × 107 genome copies/g of brain) on days 3 and 7 post-infection (p.i.), respectively. Immunohistochemistry analysis showed that ZIKV antigens were distributed in several regions of the brain, especially the dorsal hippocampus. The number of Iba1+/TMEM119+ microglia remained similar in infected versus noninfected mice, but their cell body and arborization areas significantly increased in the stratum radiatum and stratum lacunosum-moleculare layers of the dorsal hippocampus cornu ammoni (CA)1, indicating a reactive state. Ultrastructural analyses also revealed that microglia displayed increased phagocytic activities and extracellular digestion of degraded elements during infection. Mice pharmacologically depleted in microglia with PLX5622 presented a higher brain viral load compared to untreated group (2.8 × 1010 versus 8.5 × 108 genome copies/g of brain on day 10 p.i.) as well as an increased number of ZIKV antigens labeled with immunogold in the cytoplasm and endoplasmic reticulum of neurons and astrocytes indicating an enhanced viral replication. Furthermore, endosomes of astrocytes contained nanogold particles together with digested materials, suggesting a compensatory phagocytic activity upon microglial depletion. CONCLUSIONS: These results indicate that microglia are involved in the control of ZIKV replication and/or its elimination in the brain. After depletion of microglia, the removal of ZIKV-infected cells by phagocytosis could be partly compensated by astrocytes.
Asunto(s)
Encéfalo/virología , Microglía/metabolismo , Neuronas/metabolismo , Fagocitosis/fisiología , Infección por el Virus Zika/metabolismo , Animales , Encéfalo/metabolismo , Ratones , Microglía/virología , Neuronas/virologíaRESUMEN
The role of a signaling pathway through macrophage colony-stimulating factor (MCSF) and its receptor, macrophage colony-stimulating factor 1 receptor (CSF1R), during experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE) was studied by two different approaches. First, we evaluated the effect of stimulation of the MCSF/CSF1R axis before infection. Exogenous MCSF (40 µg/kg of body weight intraperitoneally [i.p.]) was administered once daily to BALB/c mice on days 4 and 2 before intranasal infection with 2,500 PFU of HSV-1. MCSF treatment significantly increased mouse survival compared to saline (50% versus 10%; P = 0.0169). On day 6 postinfection (p.i.), brain viral titers were significantly decreased, whereas beta interferon (IFN-ß) was significantly increased in mice treated with MCSF compared to mice treated with saline. The number of CD68+ (a phagocytosis marker) microglial cells was significantly increased in MCSF-treated mice compared to the saline-treated group. Secondly, we conditionally depleted CSF1R on microglial cells of CSF1R-loxP-CX3CR1-cre/ERT2 mice (in a C57BL/6 background) through induction with tamoxifen. The mice were then infected intranasally with 600,000 PFU of HSV-1. The survival rate of mice depleted of CSF1R (knockout [KO] mice) was significantly lower than that of wild-type (WT) mice (0% versus 67%). Brain viral titers and cytokine/chemokine levels were significantly higher in KO than in WT animals on day 6 p.i. Furthermore, increased infiltration of monocytes into the brains of WT mice was seen on day 6 p.i., but not in KO mice. Our results suggest that microglial cells are essential to control HSE at early stages of the disease and that the MCSF/CSF1R axis could be a therapeutic target to regulate their response to infection.IMPORTANCE Microglia appear to be one of the principal regulators of neuroinflammation in the central nervous system (CNS). An increasing number of studies have demonstrated that the activation of microglia could result in either beneficial or detrimental effects in different CNS disorders. Hence, the role of microglia during herpes simplex virus encephalitis (HSE) has not been fully characterized. Using experimental mouse models, we showed that an early activation of the MCSF/CSF1R axis improved the outcome of the disease, possibly by inducing a proliferation of microglia. In contrast, depletion of microglia before HSV-1 infection worsened the prognosis of HSE. Thus, an early microglial response followed by sustained infiltration of monocytes and T cells into the brain seem to be key components for a better clinical outcome. These data suggest that microglia could be a potential target for immunomodulatory strategies combined with antiviral therapy to better control the outcome of this devastating disease.
Asunto(s)
Encefalitis por Herpes Simple/metabolismo , Herpesvirus Humano 1/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Microglía/metabolismo , Microglía/virología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Animales , Encéfalo/virología , Sistema Nervioso Central/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Macrófagos/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Fagocitosis , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Carga ViralRESUMEN
BACKGROUND: Baloxavir is a cap-dependent inhibitor of the polymerase acid (PA) protein of influenza viruses. While appearing virologically superior to oseltamivir, baloxavir exhibits a low barrier of resistance. We sought to assess the impact of the common baloxavir-resistant I38T PA substitution on in vitro properties and virulence. METHODS: Influenza A/Quebec/144147/2009 (H1N1)pdm09 and A/Switzerland/9715293/2013 (H3N2) recombinant viruses and their I38T PA mutants were compared in single and competitive infection experiments in ST6GalI-MDCK cells and C57/BL6 mice. Virus titers in cell culture supernatants and lung homogenates were determined by virus yield assays. Ratios of wild-type (WT) and I38T mutant were assessed by digital RT-PCR. RESULTS: I38T substitution did not alter the replication kinetics of A(H1N1)pdm09 and A(H3N2) viruses. In competition experiments, a 50%:50% mixture evolved to 70%:30% (WT/mutant) for A(H1N1) and 88%:12% for A(H3N2) viruses after a single cell passage. The I38T substitution remained stable after 4 passages in vitro. In mice, the WT and its I38T mutant induced similar weight loss with comparable lung titers in both viral subtypes. The mutant virus tended to predominate over the WT in mouse competition experiments. CONCLUSION: The fitness of baloxavir-resistant I38T PA mutants appears relatively unaltered in seasonal subtypes warranting surveillance for its dissemination.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Oxazinas/farmacología , Piridinas/farmacología , ARN Polimerasa Dependiente del ARN/genética , Tiepinas/farmacología , Triazinas/farmacología , Proteínas Virales/genética , Sustitución de Aminoácidos , Animales , Antivirales/uso terapéutico , Dibenzotiepinas , Perros , Femenino , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H3N2 del Virus de la Influenza A/enzimología , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Concentración 50 Inhibidora , Pulmón/virología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Morfolinas , Mutación , Infecciones por Orthomyxoviridae/virología , Oxazinas/uso terapéutico , Fenotipo , Piridinas/uso terapéutico , Piridonas , ARN Polimerasa Dependiente del ARN/metabolismo , Tiepinas/uso terapéutico , Triazinas/uso terapéutico , Carga Viral/efectos de los fármacos , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Background: Zika virus (ZIKV) infection has been associated with prolonged viral excretion in human semen and causes testicular atrophy and infertility in 10-week-old immunodeficient mice. Methods: Male IFNAR-/- mice, knockout for type I interferon receptor, were immunized with GLS-5700, a deoxyribonucleic acid-based vaccine, before a subcutaneous ZIKV challenge with 6 × 105 plaque-forming units at 13 weeks of age. On day 28 postinfection, testes and epididymides were collected in some mice for histological and functional analyses, whereas others were mated with naive female wild-type C57BL/6J. Results: Although all mice challenged with ZIKV developed viremia, most of them were asymptomatic, showed no weight loss, and survived infection. On day 28 postinfection, none of the unvaccinated, infected mice (9 of 9) exhibited abnormal spermatozoa counts or motility. However, 33% (3 of 9) and 36% (4 of 11) of mated males from this group were infertile, from 2 independent studies. Contrarily, males from the noninfected and the vaccinated, infected groups were all fertile. On days 75 and 207 postinfection, partial recovery of fertility was observed in 66% (2 of 3) of the previously infertile males. Conclusions: This study reports the effects of ZIKV infection on male fertility in a sublethal, immunodeficient mouse model and the efficacy of GLS-5700 vaccination in preventing male infertility.
Asunto(s)
ADN/farmacología , Infertilidad Masculina/tratamiento farmacológico , Infertilidad Masculina/etiología , Infertilidad Masculina/prevención & control , Infección por el Virus Zika/complicaciones , Animales , Atrofia/etiología , Modelos Animales de Enfermedad , Epidídimo/patología , Femenino , Inmunización , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Receptor de Interferón alfa y beta/genética , Semen , Conducta Sexual Animal , Recuento de Espermatozoides , Motilidad Espermática , Espermatozoides , Testículo/patología , VacunaciónRESUMEN
We identified an influenza B isolate harboring a Gly407Ser neuraminidase substitution in an immunocompromised patient in Canada before antiviral therapy. This mutation mediated reduced susceptibility to oseltamivir, zanamivir, and peramivir, most likely by preventing interaction with the catalytic Arg374 residue. The potential emergence of such variants emphasizes the need for new antivirals.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral , Virus de la Influenza B/efectos de los fármacos , Virus de la Influenza B/enzimología , Gripe Humana/epidemiología , Neuraminidasa/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Antivirales/uso terapéutico , Canadá/epidemiología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Virus de la Influenza B/clasificación , Virus de la Influenza B/genética , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Mutación , Neuraminidasa/genética , Análisis de Secuencia de ADN , Proteínas Virales/genéticaRESUMEN
An onset of respiratory disease in a captive bachelor group (n = 3) of western lowland gorillas (Gorilla gorilla gorilla) was concomitant with peak attendance of visitors at the institution and with unwanted occurrences of food items being thrown in the gorillas' enclosure. While the condition of two individuals improved with supportive therapy and antibiotics, the third gorilla died three days following initiation of treatment. A fatal bacterial pneumonia, secondary to an infection by a human parainfluenza virus 2 (HIPV-2), was considered to be the cause of death based on histopathology, lung cultures, and reverse transcription PCR. HPIV-2 activity in the human population of the province was detected for that period, including the same viral strain. This report confirms a HPIV-2 respiratory illness and associated death in a gorilla. Clinical presentation and context suggest conspecifics were also affected and that contaminated food thrown by visitors may have been the source of infection.
Asunto(s)
Enfermedades del Simio Antropoideo/virología , Gorilla gorilla/virología , Virus de la Parainfluenza 2 Humana/aislamiento & purificación , Infecciones por Respirovirus/veterinaria , Animales , Animales de Zoológico , Enfermedades del Simio Antropoideo/mortalidad , Infecciones por Respirovirus/mortalidad , Infecciones por Respirovirus/virologíaRESUMEN
Toll-like receptors and RNA helicases are involved in the control of RNA virus infection through production of type I interferons (IFNs). To delineate the relative contributions of these signalling pathways in the innate immune response and the control of Zika virus (ZIKV) pathogenesis, the impact of a deficiency in TRIF and/or IPS-1 adaptor proteins was investigated in mice. Mice were infected intravenously with ZIKV and monitored for clinical signs for 14 days. Groups of mice were sacrificed on days 1, 3 and 7 post-infection (p.i.) and viral RNA was measured by digital droplet PCR in serum, spleen, brain and eyes. Some mice were sacrificed at 12 h p.i. for determination of the levels of IFN-α/-ß (ELISA), cytokines/chemokines (Luminex) and total/phosphorylated IRF3 and IRF7 (Western blotting). All groups of mice infected with ZIKV exhibited no clinical signs of infection. However, IPS-1-/- and TRIF-/-xIPS-1-/- mice developed higher viraemia than WT and TRIF-/- groups on days 1, 3 and 7. TRIF-/-xIPS-1-/- mice presented higher viral RNA levels in spleen, brain and eyes over time than TRIF-/-, IPS-1-/- and WT groups. At 12 h, IFN-α/-ß and cytokine/chemokine levels in spleen were significantly decreased in IPS-1-/- and TRIF-/-xIPS-1-/- compared to WT and TRIF-/-. On day 1 p.i., IFN-ß levels were significantly reduced in spleen of TRIF-/-xIPS-1-/- mice compared to all other groups. These data suggest that IPS-1 plays a more important role than TRIF in the early type I IFN response and that both IPS-1 and TRIF are involved at later stages of ZIKV infection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Inmunidad Innata , Transducción de Señal , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Encéfalo/virología , Ojo/virología , Femenino , Interferón Tipo I/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Bazo/virología , Carga Viral , Infección por el Virus Zika/virologíaRESUMEN
Human herpesviruses 6A/B (HHV-6A/B) can integrate their viral genomes in the telomeres of human chromosomes. The viral and cellular factors contributing to HHV-6A/B integration remain largely unknown, mostly due to the lack of efficient and reproducible cell culture models to study HHV-6A/B integration. In this study, we characterized the HHV-6A/B integration efficiencies in several human cell lines using two different approaches. First, after a short-term infection (5 h), cells were processed for single-cell cloning and analyzed for chromosomally integrated HHV-6A/B (ciHHV-6A/B). Second, cells were infected with HHV-6A/B and allowed to grow in bulk for 4 weeks or longer and then analyzed for the presence of ciHHV-6. Using quantitative PCR (qPCR), droplet digital PCR, and fluorescent in situ hybridization, we could demonstrate that HHV-6A/B integrated in most human cell lines tested, including telomerase-positive (HeLa, MCF-7, HCT-116, and HEK293T) and telomerase-negative cell lines (U2OS and GM847). Our results also indicate that inhibition of DNA replication, using phosphonoacetic acid, did not affect HHV-6A/B integration. Certain clones harboring ciHHV-6A/B spontaneously express viral genes and proteins. Treatment of cells with phorbol ester or histone deacetylase inhibitors triggered the expression of many viral genes, including U39, U90, and U100, without the production of infectious virus, suggesting that the tested stimuli were not sufficient to trigger full reactivation. In summary, both integration models yielded comparable results and should enable the identification of viral and cellular factors contributing to HHV-6A/B integration and the screening of drugs influencing viral gene expression, as well as the release of infectious HHV-6A/B from the integrated state.IMPORTANCE The analysis and understanding of HHV-6A/B genome integration into host DNA is currently limited due to the lack of reproducible and efficient viral integration systems. In the present study, we describe two quantitative cell culture viral integration systems. These systems can be used to define cellular and viral factors that play a role in HHV-6A/B integration. Furthermore, these systems will allow us to decipher the conditions resulting in virus gene expression and excision of the integrated viral genome resulting in reactivation.
Asunto(s)
Herpesvirus Humano 6/fisiología , Cultivo de Virus/métodos , Integración Viral , Técnicas de Cultivo de Célula/métodos , Línea Celular , Humanos , Hibridación Fluorescente in Situ , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The H275Y and E119D neuraminidase (NA) mutations constitute important molecular markers of resistance to NA inhibitors in A(H1N1) pdm09 viruses. We used reverse transcriptase-droplet digital PCR amplification (RT-ddPCR) to analyze quasi-species at codons 275 and 119 of the NA in A(H1N1) pdm09 viruses recovered from an immuncompromised patient who received oseltamivir and zanamivir therapies. RT-ddPCR assays detected and quantified H275Y and E119D mutations with an efficiency that was comparable to that of high throughput sequencing (HiSeq 2500 Illumina, San Diego, CA) technology. With its sensitivity and reproducibility, RT-ddPCR could be a reliable method for accurate detection and quantification of major NAI-resistance mutations in clinical settings. J. Med. Virol. 89:737-741, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Antivirales/uso terapéutico , Variación Genética , Subtipo H1N1 del Virus de la Influenza A/enzimología , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Neuraminidasa/genética , Reacción en Cadena de la Polimerasa/métodos , Codón , Farmacorresistencia Viral , Técnicas de Genotipaje/métodos , Humanos , Masculino , Pruebas de Sensibilidad Microbiana/métodos , Persona de Mediana Edad , Mutación Missense , Oseltamivir/uso terapéutico , Sensibilidad y Especificidad , Zanamivir/uso terapéuticoRESUMEN
The human metapneumovirus (HMPV) fusion (F) protein is the most immunodominant protein, yet subunit vaccines containing only this protein do not confer complete protection. The HMPV matrix (M) protein induces the maturation of antigen-presenting cells in vitro. The inclusion of the M protein into an F protein subunit vaccine might therefore provide an adjuvant effect. We administered the F protein twice intramuscularly, adjuvanted with alum, the M protein or both, to BALB/c mice at 3 week intervals. Three weeks after the boost, mice were infected with HMPV and monitored for 14 days. At day 5 post-challenge, pulmonary viral titres, histopathology and cytokine levels were analysed. Mice immunized with F+alum and F+M+alum generated significantly more neutralizing antibodies than mice immunized with F only [titres of 47 ± 7 (P<0.01) and 147 ± 13 (P<0.001) versus 17 ± 2]. Unlike F only [1.6 ± 0.5 × 10(3) TCID50 (g lung)(-1)], pulmonary viral titres in mice immunized with F+M and F+M+alum were undetectable. Mice immunized with F+M presented the most important reduction in pulmonary inflammation and the lowest T-helper Th2/Th1 cytokine ratio. In conclusion, addition of the HMPV-M protein to an F protein-based vaccine modulated both humoral and cellular immune responses to subsequent infection, thereby increasing the protection conferred by the vaccine.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Metapneumovirus/inmunología , Proteínas de la Matriz Viral/inmunología , Vacunas Virales/farmacología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Células Presentadoras de Antígenos , Línea Celular , Citocinas/inmunología , Humanos , Inmunidad Celular/inmunología , Inmunización/métodos , Pulmón/inmunología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Infecciones por Paramyxoviridae/inmunología , Infecciones por Paramyxoviridae/prevención & control , Infecciones por Paramyxoviridae/virología , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/prevención & control , Infecciones del Sistema Respiratorio/virología , Linfocitos T Colaboradores-Inductores , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/farmacología , Vacunas Virales/inmunologíaRESUMEN
The evolution of oseltamivir resistance mutations during selection through serial passages in animals is still poorly described. Herein, we assessed the evolution of neuraminidase (NA) and hemagglutinin (HA) genes of influenza A/WSN/33 (H1N1) and A/Victoria/3/75 (H3N2) viruses recovered from the lungs of experimentally infected BALB/c mice receiving suboptimal doses (0.05 and 1 mg/kg of body weight/day) of oseltamivir over two generations. The traditional phenotypic and genotypic methods as well as deep-sequencing analysis were used to characterize the potential selection of mutations and population dynamics of oseltamivir-resistant variants. No oseltamivir-resistant NA or HA changes were detected in the recovered A/WSN/33 viruses. However, we observed a positive selection of the I222T NA substitution in the recovered A/Victoria/3/75 viruses, with a frequency increasing over time and with an oseltamivir concentration from 4% in the initial pretherapy inoculum up to 28% after two lung passages. Although the presence of mixed I222T viral populations in mouse lungs only led to a minimal increase in oseltamivir 50% enzyme-inhibitory concentrations (IC50s) (by a mean of 5.7-fold) compared to that of the baseline virus, the expressed recombinant A/Victoria/3/75 I222T NA protein displayed a 16-fold increase in the oseltamivir IC50 level compared to that of the recombinant wild type (WT). In conclusion, the combination of serial in vivo passages under neuraminidase inhibitor (NAI) pressure and temporal deep-sequencing analysis enabled, for the first time, the identification and selection of the oseltamivir-resistant I222T NA mutation in an influenza H3N2 virus. Additional in vivo selection experiments with other antivirals and drug combinations might provide important information on the evolution of antiviral resistance in influenza viruses.
Asunto(s)
Farmacorresistencia Viral/genética , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Oseltamivir/farmacología , Proteínas Virales/genética , Animales , Antivirales/farmacología , Secuencia de Bases , Línea Celular , Perros , Inhibidores Enzimáticos/farmacología , Evolución Molecular , Hemaglutininas Virales/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Neuraminidasa/genética , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/virología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Selección Genética , Análisis de Secuencia de ARNRESUMEN
Live-Attenuated Vaccines (LAVs) stimulate robust mucosal and cellular responses and have the potential to protect against Respiratory Syncytial Virus (RSV) and Human Metapneumovirus (HMPV), the main etiologic agents of viral bronchiolitis and pneumonia in children. We inserted the RSV-F gene into an HMPV-based LAV (Metavac®) we previously validated for the protection of mice against HMPV challenge, and rescued a replicative recombinant virus (Metavac®-RSV), exposing both RSV- and HMPV-F proteins at the virion surface and expressing them in reconstructed human airway epithelium models. When administered to BALB/c mice by the intranasal route, bivalent Metavac®-RSV demonstrated its capacity to replicate with reduced lung inflammatory score and to protect against both RSV and lethal HMPV challenges in vaccinated mice while inducing strong IgG and broad RSV and HMPV neutralizing antibody responses. Altogether, our results showed the versatility of the Metavac® platform and suggested that Metavac®-RSV is a promising mucosal bivalent LAV candidate to prevent pneumovirus-induced diseases.
RESUMEN
BACKGROUND: Human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are leading pediatric pathogens. However, risk factors for severe hMPV disease remain unknown. We comparatively assessed environmental, host, and viral determinants for severe hMPV and RSV infections. METHODS: We studied a prospective cohort of >1000 children aged <3 years hospitalized in or presenting to a pediatric clinic for acute respiratory infection. We collected clinical data at enrollment and 1-month follow-up and tested nasopharyngeal secretions for respiratory viruses. Disease severity was defined as hospitalization and was also assessed with a severity score (1 point/variable) calculated on the basis of fraction of inhaled O(2) ≥ 30%, hospitalization >5 days, and pediatric intensive care unit admission. RESULTS: hMPV was identified in 58 of 305 outpatient children (19.0%) and 69 of 734 hospitalized children (9.4%), second only to RSV (48.2% and 63.6%, respectively). In multivariate regression analysis of hMPV cases, age <6 months and household crowding were associated with hospitalization. Among hospitalized patients, risk factors for severe hMPV disease were female sex, prematurity, and genotype B infection. Age <6 months, comorbidities, and household crowding were risk factors for RSV hospitalization; breast-feeding and viral coinfection were protective. Age <6 months and prematurity were associated with severe RSV cases among hospitalized children. CONCLUSIONS: hMPV and RSV severity risk factors may differ slightly. These findings will inform hMPV prevention strategies.
Asunto(s)
Metapneumovirus , Infecciones por Paramyxoviridae/patología , Infecciones por Virus Sincitial Respiratorio/patología , Virus Sincitiales Respiratorios , Factores de Edad , Preescolar , Estudios de Cohortes , Infecciones Comunitarias Adquiridas/patología , Infecciones Comunitarias Adquiridas/virología , Aglomeración , Composición Familiar , Femenino , Hospitalización , Humanos , Lactante , Recién Nacido , Recien Nacido Prematuro , Masculino , Infecciones por Paramyxoviridae/epidemiología , Infecciones por Paramyxoviridae/virología , Estudios Prospectivos , Quebec/epidemiología , Infecciones por Virus Sincitial Respiratorio/epidemiología , Infecciones por Virus Sincitial Respiratorio/virología , Factores de Riesgo , Índice de Severidad de la Enfermedad , Factores SexualesRESUMEN
Seasonal influenza A and B viruses may cause severe infections requiring therapeutic interventions. Baloxavir, the latest antiviral drug approved against those infections, targets the endonuclease activity encoded by the polymerase acidic (PA) protein. While appearing effective at cessation of viral shedding, baloxavir demonstrated a low barrier of resistance. Herein, we aimed to assess the impact of PA-I38T substitution, a major marker of baloxavir-resistance, on the fitness of contemporary influenza B viruses. Recombinant wild-type (WT) influenza B/Phuket/2073/13 (B/Yamagata/16/88-like) and B/Washington/02/19 (B/Victoria/2/87-like) viruses and their respective PA-I38T mutants were used to evaluate replication kinetics in vitro, using A549 and Calu3 cells, and ex vivo, using nasal human airway epithelium (HAE) cells. Infectivity was also assessed in guinea pigs. In the B/Washington/02/19 background, there were no major differences between the recombinant WT virus and its I38T mutant when viral replication kinetics were evaluated in human lung cell lines and HAE as well as in nasal washes of experimentally infected guinea pigs. By contrast, the I38T mutation moderately impacted the B/Phuket/2073/13 viral fitness. In conclusion, contemporary influenza B viruses that may acquire baloxavir-resistance through the PA-I38T substitution could retain a significant level of fitness, highlighting the importance of monitoring the emergence of such variant.
RESUMEN
OBJECTIVE: To develop a new method for reliable and rapid determination of the fitness of SARS-CoV-2 variants of concern. METHODS: Competition experiments between two SARS-CoV-2 variants were performed in cells of the upper (nasal human airway epithelium) and lower (Calu-3 cells) respiratory tracts followed by quantification of variant ratios by droplet digital reverse transcription (ddRT)-PCR. RESULTS: In competition experiments, the delta variant outcompeted the alpha variant in both cells of the upper and lower respiratory tracts. A 50/50% mixture of delta and omicron variants indicated a predominance of omicron in the upper respiratory tract whereas delta predominated in the lower respiratory tract. There was no evidence of recombination events between variants in competition as assessed by whole gene sequencing. CONCLUSION: Differential replication kinetics were shown between variants of concern which may explain, at least partly, the emergence and disease severity associated with new SARS-CoV-2 variants.