TRIM21 reduces H1N1-induced inflammation and apoptosis by regulating the TBK1-IRF3 signaling pathway in A549 cells.

Triple motif protein 21 (TRIM21) has an antiviral function that inhibits various viral infections. However, its role in the progress of influenza A virus (IAV) infection is unclear. In this study, we investigated the role and molecular mechanism of TRIM21 in IAV infection. RT-qPCR was used to determine the level of TRIM21 mRNA, and ELISA was used to measure the levels of IFN-α, IFN-ß, IL-6, and TNF-α. The levels of the TRIM21, NP, TBK1, IRF3, p-TBK1, and p-IRF3 proteins were estimated by Western blot. The results showed that, after IAV infection, TRIM21 was upregulated in clinical patient serum and A549 cells, and this was correlated with the IFN response. Overexpression of TRIM21 reduced IAV replication and transcription in in vitro cell experiments. TRIM21 also increased IFN-α and IFN-ß levels and decreased IL-6 and TNF-α levels in A549 cells. In addition, overexpression of TRIM21 inhibited IAV-induced apoptosis. Further experiments demonstrated that TBK1-IRF3 signaling was activated by TRIM21 and was involved in the inhibitory effect of TRIM21 on virus replication. In summary, our study suggests that TRIM21 inhibits viral replication by activating the TBK1-IRF3 signaling pathway, further inhibiting the infection process of IAV.

Isolation by multistep chromatography improves the consistency of secreted recombinant influenza neuraminidase antigens.

Recombinant protein-based approaches are ideally suited for producing vaccine antigens that are not overly abundant in viruses, such as influenza neuraminidase (NA). However, obtaining sufficient quantities of recombinant viral surface antigens remains challenging, often resulting in the use of chimeric proteins with affinity tags that can invariably impact the antigen's properties. Here, we developed multistep chromatography approaches for purifying secreted recombinant NA (rNA) antigens that are derived from recent H1N1 and H3N2 viruses and produced using insect cells. Analytical analyses showed that these isolation procedures yielded homogenous tetrameric rNA preparations with consistent specific activities that were not possible from a common immobilized metal affinity chromatography purification procedure. The use of classical chromatography improved the rNA tetramer homogeneity by removing the requirement of the N-terminal poly-histidine affinity tag that was shown to promote higher order rNA oligomer formation. In addition, these procedures reduced the specific activity variation by eliminating the exposure to Ni2+ ions and imidazole, with the latter showing pH and NA subtype dependent effects. Together, these results demonstrate that purification by multistep chromatography improves the homogeneity of secreted rNAs and eliminates the need for affinity tag-based approaches that can potentially alter the properties of these recombinant antigens.

Host Cell-dependent Modulatory Role of Ras Homolog Enriched in Brain-Like-1 (RhebL1) Protein in Influenza A/NWS/33 Virus-infected Mammalian Cells.

BACKGROUND: The Mammalian Target of Rapamycin (mTOR) signaling pathway regulates protein phosphorylation and exerts control over major cellular processes. mTOR is activated by the small G-protein Ras Homolog Enriched in Brain (Rheb), which is encoded by the Rheb1 and Rheb-like-1 (RhebL1) genes. There is currently a paucity of information on the role of RhebL1, and specifically its involvement in viral infection. In the present study we investigated the role of RhebL1 during human influenza A/NWS/33 (NWS/33) (H1N1) virus infection of rhesus monkey-kidney (LLC-MK2) cells and human type II alveolar epithelial (A549) cells. METHODS: To assess the efficiency of NWS/33 virus replication, the expression of viral nucleoprotein was examined by indirect immunofluorescence (IIF) and the viral yield by fifty percent tissue culture infectious dose assay. An RNA-mediated RNA interference approach was used to investigate the role of RhebL1 during NWS/33 infection. RhebL1 expression was evaluated by IIF, Western blotting, and enzyme-linked immunosorbent assays. A two-tailed Student's t-test was applied to evaluate differences between groups. RESULTS: RhebL1 was differentially expressed in the cell models used in this study. Silencing of the RhebL1 gene led to increased NWS/33 virus infection in A549 cells, but not in LLC-MK2 cells. Moreover, the expression of hyperphosphorylated cytokeratin 8, a marker of NWS/33 virus infection efficiency, increased in A549 cells depleted of RhebL1 but remained almost unchanged in LLC-MK2 cells. CONCLUSIONS: These are the first results showing involvement of the endogenous RhebL1 protein during viral infection. Our data suggests that RhebL1 exerts a host cell-dependent modulatory role during influenza virus infection. RhebL1 appears to be a restrictive factor against NWS/33 virus replication in A549 cells, but not in LLC-MK2.

Functional Analysis of GRSF1 in the Nuclear Export and Translation of Influenza A Virus mRNAs.

Influenza A viruses (IAV) utilize host proteins throughout their life cycle to infect and replicate in their hosts. We previously showed that host adaptive mutations in avian IAV PA help recruit host protein G-Rich RNA Sequence Binding Factor 1 (GRSF1) to the nucleoprotein (NP) 5' untranslated region (UTR), leading to the enhanced nuclear export and translation of NP mRNA. In this study, we evaluated the impact of GRSF1 in the viral life cycle. We rescued and characterized a 2009 pH1N1 virus with a mutated GRSF1 binding site in the 5' UTR of NP mRNA. Mutant viral growth was attenuated relative to pH1N1 wild-type (WT) in mammalian cells. We observed a specific reduction in the NP protein production and cytosolic accumulation of NP mRNAs, indicating a critical role of GRSF1 in the nuclear export of IAV NP mRNAs. Further, in vitro-transcribed mutated NP mRNA was translated less efficiently than WT NP mRNA in transfected cells. Together, these findings show that GRSF1 binding is important for both mRNA nuclear export and translation and affects overall IAV growth. Enhanced association of GRSF1 to NP mRNA by PA mutations leads to rapid virus growth, which could be a key process of mammalian host adaptation of IAV.

Genomic Analysis of Influenza A and B Viruses Carrying Baloxavir Resistance-Associated Substitutions Serially Passaged in Human Epithelial Cells.

Baloxavir marboxil (baloxavir) is an FDA-approved inhibitor of the influenza virus polymerase acidic (PA) protein. Here, we used next-generation sequencing to compare the genomic mutational profiles of IAV H1N1 and H3N2, and IBV wild type (WT) and mutants (MUT) viruses carrying baloxavir resistance-associated substitutions (H1N1-PA I38L, I38T, and E199D; H3N2-PA I38T; and IBV-PA I38T) during passaging in normal human bronchial epithelial (NHBE) cells. We determined the ratio of nonsynonymous to synonymous nucleotide mutations (dN/dS) and identified the location and type of amino acid (AA) substitutions that occurred at a frequency of ≥30%. We observed that IAV H1N1 WT and MUT viruses remained relatively stable during passaging. While the mutational profiles for IAV H1N1 I38L, I38T, and E199D, and IBV I38T MUTs were relatively similar after each passage compared to the respective WTs, the mutational profile of the IAV H3N2 I38T MUT was significantly different for most genes compared to H3N2 WT. Our work provides insight into how baloxavir resistance-associated substitutions may impact influenza virus evolution in natural settings. Further characterization of the potentially adaptive mutations identified in this study is needed.

Aumento de consultas en atención primaria por infección respiratoria de vías altas y fiebre coincidiendo con la epidemia de gripe (H1N1) 2009 / Increased primary care consultations for upper respiratory tract infections and fever coinciding with a wave of influenza(H1N1) 2009

Fundamento: En verano de 2009 se registró en Navarra una onda de gripe A (H1N1) 2009. Evaluar su repercusión en consultas de atención primaria con diagnóstico diferente al de gripe. Métodos: Estudiamos las consultas en atención primaria del Servicio Navarro de Salud desde el 21 de junio y al 21 de septiembre de 2009 con diagnósticos de gripe (Clasificación Internacional de Atención Primaria, código R80), síndrome febril (código A03), infección respiratoria aguda de vías altas (código R74) y bronquitis aguda (código R78), y las comparamos con las registradas en el mismo periodo en los tres años previos. Resultados: En verano de 2009 se notificaron 3417 casos de síndrome gripal (5,5 por 1.000 habitantes). Entre las semanas 27 y 31 se produjo un brote de gripe, con más de la mitad (87/160) de los frotis de pacientes con síndrome gripal positivos para el virus (H1N1) 2009 sin detectarse otros tipos de virus gripal. Coincidiendo con la onda de síndromes gripales observamos aumentos de consultas por síndrome febril e infección respiratoria de vías altas. En comparación con la media de los tres años anteriores, en el verano del 2009 se produjo un incremento del 44% en consultas por síndrome febril (de 3,6 a 5,3 por 1000: p<0,001), del 6% en consultas por infección de vías altas (de 13,2 a 14,1 por 1000; p<0,001) y del 8% en consultas por bronquitis aguda (de 6,3 a 6,9 por 1000; p=0,003). Estos diagnósticos supusieron 3,2 consultas adicionales por 1.000 habitantes atribuibles a la gripe, es decir, un 58% de consultas adicionales. Conclusiones: La gripe se acompaña de aumento en el número de consultas por síndrome febril y por infección respiratoria de vías altas(AU)

Background:Awave of influenza A(H1N1)2009 was registered in the summer of 2009. We evaluated its repercussion on primary care consultations not diagnosed as influenza. Methods: We analysed primary care consultations in the Navarre Health Service from 21 June to 21 September 2009 with a diagnosis of influenza (International Classification of Primary Care, code R80), febrile syndrome (code A03), acute upper respiratory tract infection (code R74), or acute bronchitis (code R78); these consultations were then compared with those occurring in the same period in the three previous years. Results: In the summer of 2009, 3,417 cases of influenza syndrome (5.5 per 1000 population) were reported. An flu outbreak occurred between week 27 and 31, with over the mild (87/160) of swabs from patients with influenza syndrome positive for the virus A(H1N1), with no other influenza types detected. Coinciding with the wave of influenza syndromes, we observed increases in consultations for febrile syndrome and upper respiratory tract infection. In comparison with the mean for the three previous years, in the summer of 2009 consultations for febrile syndrome increased by 44% (3.6 to 5.3 per 1000; p<0.001), consultations for upper respiratory tract infection by 6% (13.2 to 14.1 per 1000; p<0.001), and consultations for bronchitis by 8% (6.3 to 6.9 per 1000; p<0.003). These diagnoses represented 3.2 additional consultations per 1000 population attributable to influenza, that is, 58% more consultations. Conclusions: Influenza gives rise to increased primary care consultations for influenza syndrome as well as for other less important processes(AU)

Manifestações clínicas e evolução da infecção pelo vírus da influenza A (H1N1) em receptores de transplante renal / Clinical manifestations and evolution of infection by influenza A (H1N1) in kidney transplant recipients

INTRODUÇÃO: A emergência do surto pandêmico de influenza A, subtipo H1N1, em abril de 2009, representou um grande desafio para a logística de saúde pública. Embora a maioria dos pacientes infectados apresente manifestações clínicas e evolutivas muito semelhantes às observadas na influenza sazonal, um número significativo de indivíduos evolui com pneumonia e insuficiência respiratória aguda severa. O impacto da infecção pelo vírus influenza A, subtipo H1N1, em pacientes imunossuprimidos não é determinado. MÉTODOS: Neste estudo, foram analisadas a apresentação clínica e a evolução da influenza A, subtipo H1N1, em 19 receptores de transplante renal. Os pacientes receberam confirmação diagnóstica pela técnica de RT-PCR. O manejo clínico incluiu terapêutica antiviral com fosfato de oseltamivir e antibióticos. RESULTADOS: A população estudada foi predominantemente de indivíduos do sexo masculino (79 por cento), brancos (63 por cento), com idade média de 38,6 ± 17 anos e portadores de pelo menos uma comorbidade (53 por cento). A infecção por influenza A, subtipo H1N1, foi diagnosticada em média 41,6 ± 49,6 meses após o transplante. Os sintomas mais comuns foram: tosse (100 por cento), febre (84 por cento), dispneia (79 por cento) e mialgia (42 por cento). Disfunção aguda do enxerto foi observada em 42 por cento dos pacientes. Cinco pacientes (26 por cento) foram admitidos em Unidade de Terapia Intensiva, dois (10 por cento) necessitaram de suporte com ventilação invasiva e dois (10 por cento) receberam drogas vasoativas. A mortalidade foi de 10 por cento. CONCLUSÕES: A disfunção aguda do enxerto renal foi um achado frequente, e as características clínicas, laboratoriais e evolutivas foram comparáveis às da população geral.

INTRODUCTION: The emergence of the pan>demic outbreak of influenza A (H1N1) in April, 2009, represented a logistic challenge for public health. Although most infected patients presented clinical and evolutionary manifestations which were very similar to seasonal influenza, a significant number of individuals developed pneumonia and severe acute respiratory failure. The impact of influenza A (H1N1) in immunocompromised patients is not well established yet. METHODS: This study aimed to analyze the clinical presentations and evolution of influenza A (H1N1) in 19 kidney transplant recipients. Influenza A (H1N1) infection was confirmed by RT-PCR in all patients. Treatment included antiviral therapy with oseltamivir phosphate and antibiotics. RESULTS: The studied population was compounded mostly of white people (63 percent), males (79 percent), at a mean age of 38.6 ± 17 years and patients with at least one comorbidity (53 percent). Influenza A (H1N1) infection was identified 41.6 ± 49.6 months after transplantation. Common symptoms included cough (100 percent), fever (84 percent), dyspnea (79 percent), and myalgia (42 percent). Acute allograft dysfunction was observed in 42 percent of the patients. Five patients (26 percent) were admitted to the Intensive Care Unit, two (10 percent) required invasive ventilation support, and two (10 percent) required vasoactive drugs. Mortality rate was 10 percent. CONCLUSIONS: Acute renal allograft dysfunction was a common finding. Clinical, laboratory, and evolutionary characteristics were comparable to those in the general population.

Clinical Predictors of Novel Influenza A (H1N1) Infection in Korea

PURPOSE: Pandemic influenza A (H1N1) virus has spread rapidly and prompt diagnosis is needed for successful treatment and prevention of transmission. We investigated clinical predictors, validated the use of previous criteria with laboratory tests, and evaluated the clinical criteria for H1N1 infection in the Korean population. MATERIALS AND METHODS: We analyzed clinical and laboratory evaluation data from outpatient clinics at Severance Hospital in Seoul, Korea between November 11 and December 5, 2009. RESULTS: This analysis included a total of 828 patients. Of these, 372 (44.9%) patients were confirmed with H1N1 infection by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The most common and predictive symptom was cough (90.3%, OR 8.87, 95% CI 5.89-13.38) and about 40% of H1N1-positive patients were afebrile. The best predictive model of H1N1 infection was cough plus fever or myalgia. The sensitivities, specificities, positive predictive values, and negative predictive values of our suggested criteria were 73.9%, 69.5%, 66.4%, and 76.6%, respectively. CONCLUSION: Cough was the most common independent symptom in patients with laboratory-confirmed H1N1 infection, and while not perfect, the combination of cough plus fever or myalgia is suggested as clinical diagnostic criteria. Health care providers in Korea should suspect a cough without fever to be an early symptom of H1N1 infection.

Epidemiología de la gripe A (H1N1) en el mundo y en España / Epidemiology of influenza A (H1N1) worldwide and in Spain

El 11 de junio de 2009, la Organización Mundial de la Salud declaró establecida la situación de pandemiadebida a un nuevo virus influenza A (H1N1) de origen porcino. El virus empezó a producir casos de gripeen el mes de marzo en México, y a partir de mediados de abril en 6 semanas se extendió por todo el mundo.Su transmisibilidad es ligeramente superior a la de la gripe estacional; en cambio, su patogenicidad y virulenciason bajas. Los grupos más afectados han sido los niños, jóvenes y adultos de menos de 30 años. Lamortalidad se ha concentrado en las personas de 20 a 50 años.La pandemia ha producido en los países de clima templado 2 ondas epidémicas. La primera se desarrollódesde mediados de abril hasta mediados de agosto y afectó, en primer a lugar, a México, Estados Unidos yluego a España, Reino Unido, Japón y otros países del hemisferio norte. Unas semanas después, coincidiendocon el inicio de la estación gripal, afectó a los países del hemisferio sur, en especial Argentina, Chile,Australia y Nueva Zelanda, en los que concluyó a finales de septiembre u octubre.La segunda ola se ha desarrollado en el hemisferio norte, iniciándose a comienzos de septiembre en EstadosUnidos y México, y unas semanas más tarde en los países europeos; a mediados de diciembre se hadado por concluida, aunque la actividad gripal persiste. Esta segunda ola ha sido mucho más intensa que laprimera(AU)

On June 11, 2009, the World Health Organization declared an established pandemic due to a new influenzavirus A (H1N1) of swine origin. Initial cases were detected in Mexico in March and within 6 weeks thevirus had spread worldwide.The transmissibility of influenza A (H1NA) is slightly higher than that of the seasonal virus, but itspathogenicity and virulence are low. The main target groups of this new virus have been children andyoung adults under 30 years old. Mortality has affected mainly persons aged between 20 and 50 years old.In areas with temperate climates, two epidemic waves have occurred. The first one, from mid-April to mid-August, affected Mexico, the United States and, consecutively, Spain, England, Japan, and other countries inthe northern hemisphere. A few weeks later, coinciding with the beginning of the influenza season, theH1N1 epidemic started in the southern hemisphere countries, especially Argentina, Chile, Australia andNew Zealand; in these countries, the epidemic finished at the end of September or October.The second wave affected the northern hemisphere, starting in the United States and Mexico at thebeginning of September, and a few weeks later in European countries. In mid-December, this wave wasconsidered to have ended, although some influenza activity persists. The intensity of this second wave washigher compared to the first one(AU)