Characterization in vitro and in vivo of a pandemic H1N1 influenza virus from a fatal case.

Pandemic 2009 H1N1 (pH1N1) influenza viruses caused mild symptoms in most infected patients. However, a greater rate of severe disease was observed in healthy young adults and children without co-morbid conditions. Here we tested whether influenza strains displaying differential virulence could be present among circulating pH1N1 viruses. The biological properties and the genotype of viruses isolated from a patient showing mild disease (M) or from a fatal case (F), both without known co-morbid conditions were compared in vitro and in vivo. The F virus presented faster growth kinetics and stronger induction of cytokines than M virus in human alveolar lung epithelial cells. In the murine model in vivo, the F virus showed a stronger morbidity and mortality than M virus. Remarkably, a higher proportion of mice presenting infectious virus in the hearts, was found in F virus-infected animals. Altogether, the data indicate that strains of pH1N1 virus with enhanced pathogenicity circulated during the 2009 pandemic. In addition, examination of chemokine receptor 5 (CCR5) genotype, recently reported as involved in severe influenza virus disease, revealed that the F virus-infected patient was homozygous for the deleted form of CCR5 receptor (CCR5Δ32).

Severe acute respiratory syndrome coronavirus accessory proteins 6 and 9b interact in vivo.

The 3'proximal one-third of the severe acute respiratory syndrome coronavirus (SARS-CoV) genome encodes the structural proteins and eight accessory proteins, including 3a, 3b, 6, 7a, 7b, 8a, 8b and 9b, varying in length from 39 to 274aa which do not share significant homology with viral proteins of known coronaviruses. The SARS-CoV protein 6 is 63 amino acids in length and has been previously involved in virus pathogenicity and replication. To further analyze this functions, the interaction of SARS-CoV protein 6 with other viral and/or cellular factors has been analyzed during SARS-CoV infective cycle. Protein 6 immunoprecipitation from extracts of SARS-CoV infected cells and mass spectrometry analysis revealed an interaction of viral proteins 6 and 9b in biologically relevant conditions. This interaction has been reinforced by co-localization of both proteins in the cytoplasm of SARS-CoV infected cells.

Haemagglutinin D222G mutation found in a fatal case of pandemic (H1N1) flu in Tunisia.

Recently, the D222G substitution was observed in the HA of pandemic (H1N1) 2009 viruses isolated from fatal cases in several countries. We made a similar observation in one fatal case in Tunisia showing a D222G substitution in a virus isolate. The man was 47 years old and had no other subjacent pathologies or any known risk factors. He died after three days, suffering from severe respiratory symptoms of flu. The causal link of the D222G substitution in Tunisia with virulence must be verified. Further study is warranted to elucidate the intriguing relationship between the D222G substitution and severe disease. Constant molecular surveillance is important to monitor the pathogenicity of circulating strains and evaluate vaccine efficacy.

Spectrum of respiratory viruses in children with community-acquired pneumonia.

BACKGROUND: Community-acquired pneumonia (CAP) remains a significant cause for childhood morbidity worldwide. We designed a study with the objective of describing the frequency of respiratory viruses, especially rhinovirus (RV), human metapneumovirus (HMPV) and human bocavirus (HBoV) in hospitalized children with CAP. METHODS: A 6-year prospective study was conducted in children <14 years old admitted to the Pediatrics Department of the Severo Ochoa Hospital (Spain) with CAP. We studied the frequency of 16 respiratory viruses in nasopharyngeal aspirates. Clinical characteristics of respiratory syncytial virus (RSV)-only infections were compared with those of RV, HMPV and HBoV single infections. RESULTS: A viral pathogen was identified in 649 (73.4%) of 884 hospitalized children with CAP. Viral coinfections were detected in 30%. The rate of viral detection was significantly greater in infants <18 months (83%) than in older children (67%) (P < 0.001). The most frequently detected virus was RSV with 41.6% of positive patients followed by RV (26.2%), HBoV (17.8%), adenovirus (17.8%), HMPV (7%) and parainfluenza (7%). RSV was the most frequent virus in children <18 months, but RV was most common in the eldest group (P < 0.001). After stratifying by age, we found some significant differences among RSV, RV, HBoV and HMPV-associated infections. CONCLUSIONS: The high prevalence of viral infections supports the role of respiratory viruses, mainly RSV, RV, HBoV and HMPV in CAP of children requiring hospitalization. These findings help us to understand the etiologic disease burden and to guide research and public health policy.

Detection of alpha and betacoronaviruses in multiple Iberian bat species.

Bat coronaviruses (CoV) are putative precursors of the severe acute respiratory syndrome (SARS) CoV and other CoV that crossed the species barrier from zoonotic reservoirs into the human population. To determine the presence and distribution of CoV in Iberian bats, 576 individuals of 26 different bat species were captured in 13 locations in Spain. We report for the first time the presence of 14 coronaviruses in 9 Iberian bat species. Phylogenetic analysis of a conserved CoV genome region (RdRp gene) shows a wide diversity and distribution of alpha and betacoronavirus in Spain. Interestingly, although some of these viruses are related to other European BatCoV, or to Asian CoV, some of the viruses found in Spain cluster in new groups of α and ß CoV.

Variability of influenza AH1N1 infections in a neonatal unit in Spain.

We describe three positive influenza AH1N1 cases in a neonatal unit during the influenza pandemic in Spain. One term baby presented with an upper respiratory tract infection, another preterm infant with an apnea episode following nosocomial infection, and thirdly, a term infant of a mother with influenza AH1N1 had severe respiratory distress and pneumothoraces needing high-frequency ventilation.

Viral acute respiratory infections among infants visited in a rural hospital of southern Mozambique.

OBJECTIVES: To determine the epidemiology and clinical presentation of virus-associated acute respiratory infections (ARI) in Mozambican infants. METHODS: A systematic selection of nasopharyngeal aspirates (n = 333), collected from infants younger than 12 months who visited Manhiça District Hospital (southern Mozambique) with ARI during a 12 months respiratory syncitial virus surveillance, were tested for other common respiratory viruses. Four different polymerase chain reactions were used to diagnose rhinovirus (RV), influenza (Flu; A and B), adenovirus (ADV), human metapneumovirus (hMPV), parainfluenza (PIV; 1, 2, 3 and 4AB) and enterovirus (EV). RESULTS: At least one study virus was identified in more than half of the samples tested (185/333). Overall, 231 viruses were detected among 185 infants, listed in the order of prevalence: RV (26%), Flu (15%), ADV (14%), hMPV (7%), PIV (5%) and EV (3%). Acute respiratory infections (ARI) cases and viral episodes were seasonal and concentrate during the warm and the rainy season. Clinical features were similar among all study children regardless of the detection of virus, with the exception of ear discharge, which was more frequent among viral cases [6% (11/183) vs. 1% (2/144); P = 0.034]. Children with multiple viral infections had higher odds of severity such as nasal flaring and indrawing (OR = 2.7, P = 0.028 and OR = 3.8, P = 0.007, respectively) and higher odds of hospitalisation (OR = 4.42, P = 0.001, adjusted by age and sex). CONCLUSIONS: Viral ARI are frequent among infants visited in MHD. Strategies to prevent mild respiratory infections, and specially their complications, might alleviate health systems of source-limited settings.

Substitutions in position 222 of haemagglutinin of pandemic influenza A (H1N1) 2009 viruses in Spain.

BACKGROUND: A change of aspartic acid (D) to glycine (G) at position 222 in the haemagglutinin (HA) protein of pandemic influenza A (H1N1) 2009 viruses was described in Norway on November 2009 with considerable frequency in fatal and severe cases. This change was detected in other countries and was related only with severe disease. Other substitutions to glutamic acid (E) or asparagine (N) at position 222 were detected among pandemic viruses but it is unclear what implications might have in terms of severity. OBJECTIVES: To analyse the appearance of amino acid substitutions at position 222 in the HA protein of circulating viruses in Spain and to determine their relationships with the disease symptoms observed. STUDY DESIGN: Pandemic influenza A (H1N1) 2009 viruses detected in respiratory samples of 273 severe and 533 non-severe cases from different Spanish regions were selected for sequencing of a partial segment of HA1 subunit and studied to monitor substitutions at position 222. RESULTS: D222G substitution was only detected in viruses from 14 severe cases (5.12%). D222E was found in viruses from 47 severe (17.21%) and from 52 non-severe cases (9.75%). D222N occurred in viruses from 3 additional severe cases (0.37%). CONCLUSION: Appearance of D222G and D222E substitution in HA of pandemic influenza A (H1N1) viruses circulating in Spain might be related with severe respiratory disease.

The burden of infections by parainfluenza virus in hospitalized children in Spain.

We designed a prospective study to describe the clinical impact of the parainfluenza viruse (PIV) types detected in hospitalized children with respiratory tract infections from September 2008 to August 2010 in Spain. PIV infections were a significant proportion of viral respiratory detections (11.8% of cases). PIV types 3 and 4 were most commonly detected. There were clinical differences between PIV and respiratory syncytial virus infections.

Influenza pandemic (H1N1) 2009 activity during summer 2009. Effectiveness of the 2008-9 trivalent vaccine against pandemic influenza in Spain.

INTRODUCTION: The Spanish influenza surveillance system (SISS) maintained its activity during the summer of 2009 to monitor the influenza pandemic. OBJECTIVES: To describe pandemic influenza activity from May to September 2009 and to estimate the effectiveness of the 2008-9 seasonal influenza vaccine against laboratory-confirmed pandemic (H1N1) 2009 influenza. METHODS: Data from the SISS were used to identify the trend of pandemic (H1N1) 2009 influenza outside the influenza season. For the effectiveness study, we compared the vaccination status of notified cases [influenza-like illnesses (ILI) laboratory confirmed as pandemic influenza] with that of the test-negative controls. RESULTS: The first laboratory-confirmed case of the pandemic virus was notified in the system in week 20/2009. The ILI rate increased gradually in the study period, exceeding basic activity in week 38. The proportion of pandemic (H1N1) 2009 influenza viruses detected by the system represented 14% in week 20/2009 and rapidly increased to 90% in week 34. The adjusted vaccine effectiveness of the 2008-9 seasonal vaccine against laboratory-confirmed pandemic influenza was 12% (-30; 41). CONCLUSIONS: The SISS became an essential tool for pandemic monitoring in Spain. The improved SISS will provide more accurate information on influenza activity in future seasonal or pandemic waves. Using surveillance data, we could not demonstrate the effectiveness of the seasonal 2008-9 vaccine against laboratory-confirmed pandemic influenza.

Prolonged shedding of amantadine- and oseltamivir-resistant influenza A(H3N2) virus with dual mutations in an immunocompromised infant.

In this study, we report a case of multidrug-resistant influenza A(H3N2) virus isolated from an immunosuppressed patient with prolonged viral shedding. We also describe the genetic characterization of the haemagglutinin, neuraminidase and M2 influenza genes. The virus contained the substitutions E119V in neuraminidase and V27A in M2, which produce resistance to oseltamivir and adamantanes, respectively. This is the first report of this dual mutation pattern in multidrug-resistant influenza A(H3N2) virus.

Gripe pandémica H1N1 (2009). Experiencia de la red de laboratorios de gripe del sistema de vigilancia de la gripe en España (SVGE) / Pandemic influenza A (H1N1). The experience of the spanish laboratories of influenza network (ReLEG)

Existen tres tipos de virus de la gripe: A, B y C. Estos virus evo-lucionan constantemente debido a que presentan dos características principales, la primera es la falta de capacidad correctora de la poli-merasa viral que hace que se acumulen mutaciones puntuales en sus genes (deriva antigénica), y la segunda la naturaleza de su genoma formando por ocho segmentos lo que le permite el intercambio de genes entre distintos virus (salto antigénico). Esta plasticidad viral ha permitido que los virus de la gripe A sean capaces de adaptarse a diferentes hospedadores y adquirir capacidades pandémicas. El sistema de vigilancia de la gripe en España (SVGE) surgió como respuesta a la preocupación de que se produjera una pandemia, máxime después de los casos de gripe aviar detectados en el ser humano. Este sistema de vigilancia esta formado por dieciséis redes de médicos generales y pediatras centinela y diecinueve servicios de epidemiología, coordinados por el Centro Nacional de Epidemiolo-gía (CNE) y una red de dieciocho laboratorios, la red de laboratorios de Españoles de Gripe (ReLEG), coordinados por el Centro Nacio-nal de Microbiología (CNM). El objetivo de este artículo es presentar la actuación de la ReLEG durante la pandemia producida por el virus de la gripe (H1N1)2009, durante la temporada 2009-2010. La función principal de la red es la vigilancia de los virus circulantes mediante su detección y posterior caracterización genética y antigénica, incluyendo la detección de las mutaciones de resistencia que afectan a los fármacos en uso, princi-palmente el Oseltamivir(AU)

There are three types of influenza viruses: A, B, C. These viruses evolves constantly due to two main characteristics: the first one is the lack of the correction ability of the viral polymerase which causes the accumulation of single nucleotide mutations in the viral genes intro-duced by an error-prone viral RNA polymerase, (antigenic shift). The second one is the nature of their genome, formed by eight seg-ments, which allows the interchange of genes between two different viral strains (antigenic drift). This viral plasticity, has allowed to the influenza A viruses to infect new host species and to cause infections with a pandemic characteristics. The Spanish influenza surveillance system, SVGE (its Spanish acronym), arises as a response to the possibility of facing a pandemic situation, especially after the transmission of avian influenza viruses to humans. This surveillance system is formed by sixteen physician and paediatrics network, nineteen epidemiological services coordi-nated by the National Epidemiological Centre (CNE) and eighteen laboratories , the Spanish Laboratories of Influenza network (ReLEG), coordinated by the National Centre of Microbiology. The aim of this article is to show the action of the ReLEG, in the pandemic caused by the influenza virus A(H1N1) during the season 2009-2010. The main objective of this network is the surveillance of the circulating viruses by means of their detection and their subse-quent antigenic and genetic characterization, including the detection of resistance mutations against the main drugs, such as Oseltamivir(AU)

Ten years of global evolution of the human respiratory syncytial virus BA genotype with a 60-nucleotide duplication in the G protein gene.

The emergence of natural isolates of human respiratory syncytial virus group B (HRSV-B) with a 60-nucleotide (nt) duplication in the G protein gene in Buenos Aires, Argentina, in 1999 (A. Trento et al., J. Gen. Virol. 84:3115-3120, 2003) and their dissemination worldwide allowed us to use the duplicated segment as a natural tag to examine in detail the evolution of HRSV during propagation in its natural host. Viruses with the duplicated segment were all clustered in a new genotype, named BA (A. Trento et al., J. Virol. 80:975-984, 2006). To obtain information about the prevalence of these viruses in Spain, we tested for the presence of the duplicated segment in positive HRSV-B clinical samples collected at the Severo Ochoa Hospital (Madrid) during 12 consecutive epidemics (1996-1997 to 2007-2008). Viruses with the 60-nt duplication were found in 61 samples, with a high prevalence relative to the rest of B genotypes in the most recent seasons. Global phylogenetic and demographic analysis of all G sequences containing the duplication, collected across five continents up until April 2009, revealed that the prevalence of the BA genotype increased gradually until 2004-2005, despite its rapid dissemination worldwide. After that date and coinciding with a bottleneck effect on the population size, a relatively new BA lineage (BA-IV) replaced all other group B viruses, suggesting further adaptation of the BA genotype to its natural host.

Role of rhinovirus C respiratory infections in sick and healthy children in Spain.

BACKGROUND: Recently a new genogroup of human rhinovirus (HRV) has been described and named HRV-C. The relative importance of HRV-C in viral respiratory tract illnesses is unknown. OBJECTIVE: We looked for HRV-C in pediatric patients with respiratory tract infections to determine the incidence of HRV-C and its role in sick and healthy children. We describe the clinical differences associated with HRV-C infections and other HRV genogroups. PATIENTS AND METHODS: From January 2004 to December 2008, a prospective study was conducted in children younger than 14 years who were admitted with respiratory infection to the Pediatrics Department of the Severo Ochoa Hospital in Madrid, Spain. Specimens of nasopharyngeal aspirate were taken for virologic study with polymerase chain reaction, and clinical data were recorded. HRV specimens were genotyped. We studied the frequency of HRV-C infections, the clinical course of these patients and the differences with other HRV genogroups (HRV-A and HRV-B). Presence of HRV-C was also studied in a group of healthy children. RESULTS: HRV was detected in 424 of 1555 episodes of illness (27.2%) and in 26 of 211 healthy children (12.3%) (P < 0.001). We amplified at random 248 of them (227 hospitalized children and 21 healthy children): 132 (53.2%) had HRV-A, 28 (11.2%) had HRV-B, and 88 (35.4%) HRV-C. HRV-C infections were associated with asthma, recurrent wheezing, and bronchiolitis but were not significantly different from the HRV-A genogroup. Nevertheless, significant clinical differences were observed between the HRV-B genogroup and the other groups: more frequent infiltrate on chest radiograph (P = 0.017), fever (P = 0.052), diagnosis of pneumonia (P = 0.01), and antibiotic treatment (P = 0.004). CONCLUSIONS: HRV-C infections were frequent in hospitalized children with respiratory diseases and were associated with asthma, recurrent wheezing, and bronchiolitis. No clinical differences were found with the HRV-A group: HRV-B group had clinical differences with both the other groups.

Development and implementation of influenza a virus subtyping and detection of genotypic resistance to neuraminidase inhibitors.

Influenza virus hemagglutinin and neuraminidase, surface glycoproteins with an essential role in viral pathogenesis, are important antigen determinants and essential markers for epidemiological surveillance. Neuraminidase is also a suitable target for designing antiviral drugs. The introduction into clinical practice of neuraminidase inhibitors and the development of random point mutations have increased the emergence of drug-resistant viruses. A universal RT nested PCR-based system has been developed for subtyping H1, H3, N1 and N2, in influenza A viruses of human or animal origin. The subsequent sequencing and analysis of the hemagglutinin and neuraminidase templates reveal antigenic and receptor binding changes in the HA1 subunit and mutations of clinical relevance concerning resistance to neuraminidase inhibitors. The specificity and sensitivity of the method were evaluated using 113 influenza A isolates, 105 influenza A positive respiratory samples obtained from patients and 29 prototype strains of both human and animal origin. The resulting analytical sensitivity of the subtyping techniques is one to at least 100 molecules of cloned DNA product in a final reaction volume of 50 microl. In the course of implementing the method, two H1N1 isolates with the H274Y mutation in the neuraminidase segment have been detected and their molecular features analyzed. The emergence of influenza virus resistance makes the neuraminidase genetic characterization and surveillance activities to detect antiviral resistance necessary.

[Pandemic influenza A(H1N1): the experience of the Spanish Laboratories of Influenza Network (ReLEG)]. / Gripe pandémica H1N1 (2009): Experiencia de la Red de Laboratorios de Gripe del Sistema de Vigilancia de la Gripe en España (SVGE).

There are three types of influenza viruses: A, B, C. These viruses evolves constantly due to two main characteristics: the first one is the lack of the correction ability of the viral polymerase which causes the accumulation of single nucleotide mutations in the viral genes introduced by an error-prone viral RNA polymerase, (antigenic shift). The second one is the nature of their genome, formed by eight segments, which allows the interchange of genes between two different viral strains (antigenic drift). This viral plasticity, has allowed to the influenza A viruses to infect new host species and to cause infections with a pandemic characteristics. The Spanish influenza surveillance system, SVGE (its Spanish acronym), arises as a response to the possibility of facing a pandemic situation, especially after the transmission of avian influenza viruses to humans. This surveillance system is formed by sixteen physician and paediatrics network, nineteen epidemiological services coordinated by the National Epidemiological Centre (CNE) and eighteen laboratories , the Spanish Laboratories of Influenza network (ReLEG), coordinated by the National Centre of Microbiology. The aim of this article is to show the action of the ReLEG, in the pandemic caused by the influenza virus A(H1N1) during the season 2009-2010. The main objective of this network is the surveillance of the circulating viruses by means of their detection and their subsequent antigenic and genetic characterization, including the detection of resistance mutations against the main drugs, such as Oseltamivir.

Role of rhinovirus C in apparently life-threatening events in infants, Spain.

To assess whether infants hospitalized after an apparently life-threatening event had an associated respiratory virus infection, we analyzed nasopharyngeal aspirates from 16 patients. Nine of 11 infants with positive virus results were infected by rhinoviruses. We detected the new genogroup of rhinovirus C in 6 aspirates.

Attenuated strains of influenza A viruses do not induce degradation of RNA polymerase II.

We have previously shown that infection with laboratory-passaged strains of influenza virus causes both specific degradation of the largest subunit of the RNA polymerase II complex (RNAP II) and inhibition of host cell transcription. When infection with natural human and avian isolates belonging to different antigenic subtypes was examined, we observed that all of these viruses efficiently induce the proteolytic process. To evaluate whether this process is a general feature of nonattenuated viruses, we studied the behavior of the influenza virus strains A/PR8/8/34 (PR8) and the cold-adapted A/Ann Arbor/6/60 (AA), which are currently used as the donor strains for vaccine seeds due to their attenuated phenotype. We have observed that upon infection with these strains, degradation of the RNAP II does not occur. Moreover, by runoff experiments we observe that PR8 has a reduced ability to inhibit cellular mRNA transcription. In addition, a hypervirulent PR8 (hvPR8) variant that multiplies much faster than standard PR8 (lvPR8) in infected cells and is more virulent in mice than the parental PR8 virus, efficiently induces RNAP II degradation. Studies with reassortant viruses containing defined genome segments of both hvPR8 and lvPR8 indicate that PA and PB2 subunits individually contribute to the ability of influenza virus to degrade the RNAP II. In addition, recently it has been reported that the inclusion of PA or PB2 from hvPR8 in lvPR8 recombinant viruses, highly increases their pathogenicity. Together, the data indicate that the capacity of the influenza virus to degrade RNAP II and inhibit the host cell transcription machinery is a feature of influenza A viruses that might contribute to their virulence.

[Microbiological diagnosis of viral respiratory infections]. / Diagnóstico microbiológico de las infecciones virales respiratorias.

Acute respiratory infection is the most common disease occurring over a person's lifetime, with etiological variations determined mainly by age, environmental circumstances, the healthcare setting, and the underlying pathology. More than 200 different viruses distributed in six viral families have been implicated in the pathogenesis of respiratory tract infection. These facts are generating an increasing diagnostic demand that should be incorporated into the healthcare setting without delay. To meet this demand, the Spanish Society of Infectious Diseases and Clinical Microbiology has updated its Standard Procedure for the microbiological diagnosis of viral respiratory infection. This document contains an update primarily of infections caused by influenza viruses, and secondarily, infections due to other conventional and emerging respiratory viruses. In all cases, the methods for direct virological diagnosis (cell culture, and detection of antigens and nucleic acid) are reviewed, with special reference to techniques for molecular detection and genetic characterization.

Diagnóstico microbiológico de las infecciones virales respiratorias / Microbiological diagnosis of viral respiratory infections

La infección respiratoria aguda es la enfermedad más frecuente a lo largo de toda la vida del ser humano, con una variación en cuanto a su etiología condicionada, fundamentalmente, por la edad, las circunstancias medioambientales, el ámbito asistencial y la enfermedad de base. Se han identificado más de 200 virus diferentes distribuidos en seis familias implicados en la patogenia de las infecciones del tracto respiratorio. Estos hechos generan una demanda diagnóstica, cuya incorporación al ámbito asistencial no debe ser retrasada. Consciente de ello la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica ha redactado un procedimiento sobre diagnóstico microbiológico de las infecciones virales respiratorias. En éste se efectúa una puesta al día de las infecciones debidas, en primer término, a los virus de la gripe y, en segundo lugar, a otros virus respiratorios convencionales y emergentes. En todos los casos se revisan los métodos de diagnóstico virológico directo (cultivo celular, detección de antígenos y de ácido nucleico), con particular referencia a las técnicas de detección molecular y de caracterización genética (AU)

Acute respiratory infection is the most common disease occurring over a person's lifetime, with etiological variations determined mainly by age, environmental circumstances, the healthcare setting, and the underlying pathology. More than 200 different viruses distributed in six viral families have been implicated in the pathogenesis of respiratory tract infection. These facts are generating an increasing diagnostic demand that should be incorporated into the healthcare setting without delay. To meet this demand, the Spanish Society of Infectious Diseases and Clinical Microbiology has updated its Standard Procedure for the microbiological diagnosis of viral respiratory infection. This document contains an update primarily of infections caused by influenza viruses, and secondarily, infections due to other conventional and emerging respiratory viruses. In all cases, the methods for direct virological diagnosis (cell culture, and detection of antigens and nucleic acid) are reviewed, with special reference to techniques for molecular detection and genetic characterization (AU)