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.

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.

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.

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.

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.

Severe acute respiratory syndrome coronavirus protein 6 is required for optimal replication.

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes several accessory proteins of unknown function. One of these proteins, protein 6 (p6), which is encoded by ORF6, enhances virus replication when introduced into a heterologous murine coronavirus (mouse hepatitis virus [MHV]) but is not essential for optimal SARS-CoV replication after infection at a relatively high multiplicity of infection (MOI). Here, we reconcile these apparently conflicting results by showing that p6 enhances SARS-CoV replication to nearly the same extent as when expressed in the context of MHV if cells are infected at a low MOI and accelerates disease in mice transgenic for the human SARS-CoV receptor.

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.

Synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast Saccharomyces cerevisiae.

Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 nucleocapsid proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus nucleocapsid proteins expression (20-24 mg l(-1) of yeast culture) was obtained. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. These structures contained host RNA, which was resistant to RNase treatment. The nucleocapsid proteins were stable in yeast and were easily purified by caesium chloride gradient ultracentrifugation. Therefore, this system proved to be simple, efficient and cost-effective, suitable for high-level production of parainfluenza virus nucleocapsids as nucleocapsid-like particles. When used as coating antigens in an indirect ELISA, the recombinant N proteins reacted with sera of patients infected with HPIV1 or 3. Serological assays to detect HPIV-specific antibodies could be designed on this basis.

Human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection.

The main objective of our study was to determine the frequency of human bocavirus (HBoV) detection in asymptomatic children and to compare it with that in children hospitalized because of respiratory infection. HBoV was detected in 5% of 116 healthy children versus 17% of 908 hospitalized children. HBoV can be detected in healthy children but with a significantly lower frequency than in ill children.

Clinical characteristics of human bocavirus infections compared with other respiratory viruses in Spanish children.

BACKGROUND: Human bocavirus (HBoV) can be found in a substantial proportion of children with respiratory tract diseases. The relative importance of HBoV in viral respiratory tract illnesses is not yet well known. OBJECTIVE: In this study, we looked for HBoV in pediatric patients to determine the incidence of HBoV as single infection and compared it with other commonly found respiratory viruses to describe the clinical differences associated with HBoV infections in children. PATIENTS AND METHODS: A prospective study was conducted on children less than 14 years old, admitted with respiratory infection from September 2005 to August 2007 to the Pediatrics Department of the Severo Ochoa Hospital, Madrid, Spain. We studied the frequency of HBoV and 15 other respiratory viruses in nasopharyngeal aspirates and compared the clinical course of the infections caused by HBoV with those caused by other common respiratory viruses. RESULTS: Positive results were confirmed in 435 (61.2%) of the 710 children studied. A single virus was detected in 308 patients. HBoV was found in 99 (13.9%) samples, but it was recovered as a single virus in only 35. Most of patients with HBoV infection (75%) were aged < or =26 months. The most common clinical diagnosis was recurrent wheezing (53%), followed by bronchiolitis (32%). Clinical differences were observed between HBoV and respiratory syncytial virus (RSV) infections (children were older and bronchiolitis less frequent), adenovirus (fever less frequent in HBoV group), and rhinovirus-associated infections (less hypoxia in HBoV group). CONCLUSIONS: HBoV was the fourth most frequent single virus after RSV, rhinovirus, and adenovirus in children hospitalized because of respiratory infection. It was associated with recurrent wheezing and bronchiolitis showing a different clinical course from other virus in terms of diagnosis, fever, and age.

Multiple simultaneous viral infections in infants with acute respiratory tract infections in Spain.

BACKGROUND: The clinical significance of the presence of more than one type of virus in the respiratory specimens of children with respiratory infections is not clear. OBJECTIVES: To describe the clinical characteristics of multiple viral infections versus single infection by respiratory syncytial virus (RSV) in hospitalized infants. STUDY DESIGN: This is a prospective study conducted in all infants under 2 years of age admitted for acute respiratory infection (September 2000-June 2003) in a secondary teaching hospital. Virological diagnosis was made by two different multiplex reverse transcription-nested polymerase chain reaction (RT-PCR) assays in nasopharyngeal aspirates. We describe the clinical characteristics of the patients with multiple viral infections and compare them to a group of 86 randomly selected patients infected only with RSV. RESULTS: 749 specimens taken were analyzed. Respiratory viruses were detected in 65.9% of the samples. 86 children had multiple viral infections (17.4% of all positive specimens). The most frequent clinical diagnosis in this group was recurrent wheezing in 44% and bronchiolitis in 52%. Fever was significantly more frequent (p<0.001), hospital stays were longer (p=0.05), and antibiotic treatment was used more (p=0.03) in infants with multiple viral infections than in the RSV-infected group. CONCLUSIONS: Multiple viral infections are frequent in hospitalized children with respiratory tract disease (17.4%). Multiple viral infections are linked to higher fever, longer hospital stays and more frequent use of antibiotics than in the case of infants with single RSV infections.

Role of rhinovirus in hospitalized infants with respiratory tract infections in Spain.

BACKGROUND: Rhinovirus is a recognized cause of common cold, proven to cause asthma exacerbations in children. In Spain, no description exists, as yet, as to the degree of burden rhinovirus infections represent among hospitalized infants. Our aim was to describe rhinovirus infections in hospitalized children, under 2 years of age, and to compare these with patients infected with respiratory syncytial virus (RSV). PATIENTS AND METHODS: The prospective study was performed between September 2003 and July 2005, in children <2 years of age, admitted at the Severo Ochoa Hospital (Leganés, Madrid) with fever or respiratory tract infection and with positive rhinovirus detection in the nasopharyngeal aspirate samples. Virologic diagnosis was made by multiplex reverse transcription-polymerase chain reaction and for some virus by direct immunofluorescent assay in nasopharyngeal samples. Demographic and clinical data of those patients with rhinovirus infection were described and compared with a group of 86 patients, infected only with RSV, randomly selected from the same population. RESULTS: We detected 85 children admitted to hospital with rhinovirus infection. Rhinovirus was the cause of 25% of all admissions, among the total of 340 under 2-year olds diagnosed with fever or respiratory tract infection. Rhinovirus was the second viral agent identified, after RSV. Clinical diagnosis was recurrent wheezing in 48.2%; bronchiolitis in 36.5%; and pneumonia in 3.5%. Fever was present in 60% of the patients. Radiologic infiltrates were found in 22.4% of the children. In 50.6% of the infants, oxygen saturation under 95% was detected, at the time of admission. Hypoxia was present in RSV-infected children more frequently (P = 0.005). Also, in this group, final diagnosis was, most frequently, bronchiolitis (P = 0.0001), and rhinovirus-infected patients were most frequently males (P = 0.004). CONCLUSIONS: Rhinovirus was detected in hospitalized infants with respiratory tract disease and was the second most common virus after RSV. In our experience, it was the second etiologic agent associated with recurrent wheezing in hospitalized children, under the age of 2 years.

High incidence of human bocavirus infection in children in Spain.

BACKGROUND: The newly identified human bocavirus (HBoV), a member of the Parvoviridae family, has been associated to low respiratory tract infections in young children. OBJECTIVES: To present the epidemiological profile and the main clinical characteristics showed by children infected with this virus in Spain. STUDY DESIGN: We have studied the incidence of HBoV and other 15 respiratory viruses in 917 nasopharyngeal aspirates taken from 730 infants and children under age of 14 with acute lower respiratory tract infection from September-04 to August-06. RESULTS: HBoV was detected in 123 samples (13.4%) showing a seasonal distribution with November and December as the peak months. Out of the 558 samples which rendered a positive result for at least one of the virus tested, HBoV (22%) ranked fourth behind respiratory syncytial virus (181, 32%), adenoviruses (155, 28%) and rhinoviruses (136, 24%). Co-infections with HBoV and other respiratory viruses were detected in 74 out of 123 HBoV-positive specimens (60%). In addition, HBoV was also found in stool and, for the first time, in urine samples. CONCLUSIONS: Results obtained provide further evidence that HBoV is involved in acute lower respiratory tract infections. HBoV-associated disease should not be limited to the respiratory tract.

Oligonucleotide microarray design for detection and serotyping of human respiratory adenoviruses by using a virtual amplicon retrieval software.

The rapid development of new molecular biology methods has improved infectious disease diagnosis, which is increasingly important to clinical management and public health. A wide variety of new methods which are more specific, sensitive and robust, such as combination of PCR and microarray technology, has gradually replaced the conventional assays usually used in routine diagnosis. Both methods have the advantage of speed and sensitivity but tend to be expensive and technically demanding. Therefore, it is necessary to develop more simple assays that could be available for all diagnostic laboratories. To this aim, a simple microarray assay for detection and typing of adenoviruses causing acute respiratory disease in humans was developed. The absence of effective therapeutic or alternative prophylactic treatment for this infection makes essential its rapid diagnosis to implement fast control measures. Moreover, the family Adenoviridae includes numerous serotype groups and constitutes an ideal model system to develop diagnostic methods for other human pathogens. For their precise identification, an amplicon retrieval software that simplifies and accelerates the processing of all sequences necessary to perform this type of experiments has also been developed. This new technology was applied for the simultaneous detection and serotyping of acute respiratory disease-causing adenoviruses in laboratory and clinical samples with efficiency and accuracy.

Respiratory syncytial virus group A and B genotypes and disease severity among Cuban children.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of serious lower tract infections in infants. Comorbid conditions such as chronic diseases and prematurity have been associated with greater severity illness, but virus genotypes and disease severity is still unknown. METHODS: Forty selected strains of RSV group A and B from Cuban infants with acute respiratory disease (ARD) over five seasons were studied. Viral RNA was extracted and polymerase chain reaction (PCR) was carried out using direct primers directed to parts of the nucleoprotein (N) and fusion (F) genes, respectively. Amplicons were digested using restriction fragment length polymorphism (RFLP) to define the association between virus and disease severity. Disease severity was assessed as very mild, mild, moderate, and severe. RESULTS: Three of six known N genotypes were detected. NP4 and NP3 were found more frequently; moreover, it was difficult to establish a relationship between N genotypes and disease severity. Five genotypes in F gene were found: F1, F2, F5, F9 and F11; F9 and F11 were associated with very mild disease, but F1 genotype appears to be associated with moderate to severe disease. CONCLUSIONS: At least five combinations of N and F genotypes circulated in the studied infants in Cuba. Patients with F1NP4 genotype showed moderate to severe disease. Relationship between genotypes and disease severity was established.

Implementation of the community network of reference laboratories for human influenza in Europe.

BACKGROUND: The increased need for accurate influenza laboratory surveillance data in the European Union required formalisation of the existing network of collaborating national influenza reference laboratories participating in the European Influenza Surveillance Scheme (EISS). OBJECTIVE: To establish a Community Network of Reference Laboratories for Human Influenza in Europe (CNRL). METHODS: Virologists in EISS defined the objective and tasks of the CNRL. Performance of the laboratories in the tasks was monitored by questionnaire-based inventories and quality control assessments (QCA). Subsequently, actions were defined to improve the performance of the CNRL. RESULTS: The CNRL started in April 2003 and included as of May 2004 32 laboratories in 24 European countries. The objective is to provide high quality reference services for human influenza surveillance, early warning and pandemic preparedness in Europe. The defined basic tasks are direct detection, culture, typing, subtyping and strain characterisation of influenza virus, diagnostic influenza serology and storage of clinical specimens and virus isolates. The questionnaire-based inventories and QCAs revealed that the majority of CNRL laboratories perform well in most of the basic tasks, although improvements are needed in certain areas of virus testing. Therefore, task groups have been established to further improve the methods used in the network. The CNRL has proven its usefulness during the 2003-2004 season by the reporting of accurate data concerning the flu epidemic caused by A/Fujian/411/2002 (H3N2)-like viruses and by the rapid sharing of information, protocols and reagents during the A(H5N1) and A(H7N3) epizootics in Asia and Canada. CONCLUSION: EISS has established a functioning Community Network of Reference Laboratories for Human Influenza in Europe and laid the foundation for further enhancement and collaborations. Important next steps include improving the laboratories to carry out all basic tasks and collaboration with the European Centre for Disease Prevention and Control.

Two RT-PCR based assays to detect human metapneumovirus in nasopharyngeal aspirates.

Two sensitive and specific RT-PCR assays were standardised for testing the presence of human metapneumovirus. A total of 300 nasopharyngeal aspirates collected from infants suffering from bronchiolitis since October 2000 to June 2003 and shown previously as negative to common respiratory viruses were examined. Matrix and polymerase viral genes, which show a low rate of variation, were chosen to design amplification assays to ensure that any genotype of the human metapneumovirus could be detected. A RT-PCR followed by a reverse line blotting hybridisation was developed for viral polymerase gene. For the matrix gene, after the RT-PCR assay, a subsequent nested PCR was carried out. Both assays had similar sensitivity, equivalent to 0.1 TCID50 of human metapneumovirus strain NL/1/99 which was used as the positive control. The human metapneumovirus was present in 16.6% of the specimens studied. The approaches described below are not only a robust method for rapid diagnosis of the human metapneumovirus, but also to establish an etiological surveillance tool for epidemiological studies. Based on the results obtained, human metapneumovirus infections in Madrid followed a seasonal pattern, with most of the infections occurring between February and April.