Short report: human metapneumovirus in children with influenza-like illness in Yucatan, Mexico

The present study suggests that human metapneumovirus (hMPV) is an important cause of community acquired respiratory infections in children. We report the detection of hMPV in a pediatric population with influenza-like illness in the subtropical area of Yucatan in Mexico. Our data also shows that hMPV circulates in the community with other respiratory pathogens.

Generation of candidate human influenza vaccine strains in cell culture: rehearsing the European response to an H7N1 pandemic threat

Background: Although H5N1 avian influenza viruses pose the most obvious imminent pandemic threat, there have been several recent zoonotic incidents involving transmission of H7 viruses to humans. Vaccines are the primary public health defense against pandemics, but reliance on embryonated chickens eggs to propagate vaccine and logistic problems posed by the use of new technology may slow our ability to respond rapidly in a pandemic situation. Objectives: We sought to generate an H7 candidate vaccine virus suitable for administration to humans whose generation and amplification avoided the use of eggs. Methods: We generated a suitable H7 vaccine virus by reverse genetics. This virus, known as RD3, comprises the internal genes of A/Puerto Rico/8/34 with surface antigens of the highly pathogenic avian strain A/Chicken/Italy/13474/99 (H7N1). The multi-basic amino acid site in the HA gene, associated with high pathogenicity in chickens, was removed. Results: The HA modification did not alter the antigenicity of the virus and the resultant single basic motif was stably retained following several passages in Vero and PER. C6 cells. RD3 was attenuated for growth in embryonated eggs, chickens, and ferrets. RD3 induced an antibody response in infected animals reactive against both the homologous virus and other H7 influenza viruses associated with recent infection by H7 viruses in humans. Conclusions: This is the first report of a candidate H7 vaccine virus for use in humans generated by reverse genetics and propagated entirely in mammalian tissue culture. The vaccine has potential use against a wide range of H7 strains.

Variation in the ability of human influenza A viruses to induce and inhibit the IFN-beta pathway

We investigated the ability of a selection of human influenza A viruses, including recent clinical isolates, to induce IFN-beta production in cultured cell lines. In contrast to the well-characterized laboratory strain A/PR/8/34, several, but not all, recent isolates of H3N2 viruses resulted in moderate IFN-beta stimulation. Through the generation of recombinant viruses, we were able to show that this is not due to a loss of the ability of the NS1 genes to suppress IFN-beta induction; indeed, the NS1 genes behaved similarly with respect to their abilities to block dsRNA signaling. Interestingly, replication of A/Sydney/5/97 virus was less Susceptible to pre-treatment with IFN-alpha than the other viruses. In contrast to the universal effect on dsRNA signaling, we noted differences in the effect of NS1 proteins on expression of interferon stimulated genes and also genes induced by a distinct pathway. The majority of NS1 proteins blocked expression From both IFN-dependent and TNF-dependent promoters by an apparent post-transcriptional mechanism. The NS1 gene of A/PR/8/34 NS1 did not confer these blocks. We noted striking differences in the Cellular localization of different influenza A virus NS1 proteins during infection, which might explain differences in biological activity. (C) 2005 Elsevier Inc. All rights reserved.

Infection of human airway epithelium by human and avian strains of influenza a virus

We describe the characterization of influenza A virus infection of an established in vitro model of human pseudostratified mucociliary airway epithelium (HAE). Sialic acid receptors for both human and avian viruses, alpha-2,6- and alpha-2,3-linked sialic acids, respectively, were detected on the HAE cell surface, and their distribution accurately reflected that in human tracheobronchial tissue. Nonciliated cells present a higher proportion of alpha-2,6-linked sialic acid, while ciliated cells possess both sialic acid linkages. Although we found that human influenza viruses infected both ciliated and nonciliated cell types in the first round of infection, recent human H3N2 viruses infected a higher proportion of nonciliated cells in HAE than a 1968 pandemic-era human virus, which infected proportionally more ciliated cells. In contrast, avian influenza viruses exclusively infected ciliated cells. Although a broad-range neuraminidase abolished infection of HAE by human parainfluenza virus type 3, this treatment did not significantly affect infection by influenza viruses. All human viruses replicated efficiently in HAE, leading to accumulation of nascent virus released from the apical surface between 6 and 24 h postinfection with a low multiplicity of infection. Avian influenza A viruses also infected HAE, but spread was limited compared to that of human viruses. The nonciliated cell tropism of recent human H3N2 viruses reflects a preference for the sialic acid linkages displayed on these cell types and suggests a drift in the receptor binding phenotype of the H3 hemagglutinin protein as it evolves in humans away from its avian virus precursor.

Characterization of recombinant influenza B viruses with key neuraminidase inhibitor resistance mutations

Objectives and methods: An influenza B virus plasmid-based rescue system was used to introduce site-specific mutations, previously observed in neuraminidase (NA) inhibitor-resistant viruses, into the NA protein of six recombinant viruses. Three mutations observed only among in vitro selected zanamivir-resistant influenza A mutants were introduced into the B/Beijing/1/87 virus NA protein, to change residue E116 to glycine, alanine or aspartic acid. Residue E116 was also mutated to valine, a mutation found in the clinic among oseltamivir-resistant viruses. An arginine to lysine change at position 291 (292 N2 numbering) mimicked that seen frequently in influenza A N2 clinical isolates resistant to oseltamivir. Similarly, an arginine to lysine change at position 149 (152 in N2 numbering) was made to reproduce the change found in the only reported zanamivir-resistant clinical isolate of influenza B virus. In vitro selection and prolonged treatment in the clinic leads to resistance pathways that require compensatory mutations in the haemagglutinin gene, but these appear not to be important for mutants isolated from immunocompetent patients. The reverse genetics system was therefore used to generate mutants containing only the NA mutation. Results and conclusions: With the exception of a virus containing the E116G mutation, mutant viruses were attenuated to different levels in comparison with wild-type virus. This attenuation was a result of altered NA activity or stability depending on the introduced mutation. Mutant viruses displayed increased resistance to zanamivir, oseltamivir and peramivir, with certain viruses displaying cross-resistance to all three drugs.

The M1 matrix protein controls the filamentous phenotype of influenza A virus

We show that most isolates of influenza A induce filamentous changes in infected cells in contrast to A/WSN/33 and A/PR8/34 strains which have undergone extensive laboratory passage and are mouse-adapted. Using reverse genetics, we created recombinant viruses in the naturally filamentous genetic background of A/Victoria/3/75 and established that this property is regulated by the M1 protein sequence, but that the phenotype is complex and several residues are involved. The filamentous phenotype was lost when the amino acid at position 41 was switched from A to V, at the same time, this recombinant virus also became insensitive to the antibody 14C2. On the other hand, the filamentous phenotype could be fully transferred to a virus containing RNA segment 7 of the A/WSN/33 virus by a combination of three mutations in both the amino and carboxy regions of the M1 protein. This observation suggests that an interaction among these regions of M1 may occur during assembly. (C) 2004 Elsevier Inc. All rights reserved.

Restrictions to the adaptation of influenza A virus H5 hemagglutinin to the human host

The binding specificities of a panel of avian influenza virus subtype H5 hemagglutinin (RA) proteins bearing mutations at key residues in the receptor binding site were investigated. The results demonstrate that two simultaneous mutations in the receptor binding site resulted in H5 RA binding in a pattern similar to that shown by human viruses. Coexpression of the ion channel protein, M2, from most avian and human strains tested protected H5 RA conformation during trafficking, indicating that no genetic barrier to the reassortment of the H5 surface antigen gene with internal genes of human viruses existed at this level.

Reduced incorporation of the influenza B virus BM2 protein in virus particles decreases infectivity

BM2 is the fourth integral membrane protein encoded by the influenza B virus genome. It is synthesized late in infection and transported to the plasma membrane from where it is subsequently incorporated into progeny virus particles. It has recently been reported that BM2 has ion channel activity and may be the functional homologue of the influenza A virus M2 protein acting as an ion channel involved in viral entry. Using a reverse genetic approach it was not possible to recover virus which lacked BM2. A recombinant influenza B virus was generated in which the BM2 AUG initiation codon was mutated to GUG. This decreased the efficiency of translation of BM2 protein such that progeny virions contained only 1/8 the amount of BM2 seen in wild-type virus. The reduction in BM2 incorporation resulted in a reduction in infectivity although there was no concomitant decrease in the numbers of virions released from the infected cells. These data imply that the incorporation of sufficient BM2 protein into influenza B virions is required for infectivity of the virus particles. (C) 2004 Elsevier Inc. All rights reserved.

Changes in in vitro susceptibility of influenza A H3N2 viruses to a neuraminidase inhibitor drug during evolution in the human host

Objectives: Influenza A H3N2 viruses isolated recently have characteristic receptor binding properties that may decrease susceptibility to neuraminidase inhibitor drugs. A panel of clinical isolates and recombinant viruses generated by reverse genetics were characterized and tested for susceptibility to zanamivir. Methods: Plaque reduction assays and neuraminidase enzyme inhibition assays were used to assess susceptibility to zanamivir. Receptor binding properties of the viruses were characterized by differential agglutination of red blood cells (RBCs) from different species. Sequence analysis of the haemagglutinin (HA) and neuraminidase (NA) genes was carried out. Results: Characterization of a panel of H3N2 clinical isolates from 1968 to 2000 showed a gradual decrease in agglutination of chicken and guinea pig RBCs over time, although all isolates could agglutinate turkey RBCs equally. Sequence analysis of the HA and NA genes identified mutations in conserved residues of the HA1 receptor binding site, in particular Leu-226 --> Ile-226/Val-226, and modification of potential glycosylation site motifs. This may be indicative of changes in virus binding to sialic acid (SA) receptors in recent years. Although recent isolates had reduced susceptibility to zanamivir in MDCK cell based plaque reduction assays, no difference was found in an NA enzyme-inhibition assay. Assays with recombinant isogenic viruses showed that the recent HA, but not the NA, conferred reduced susceptibility to zanamivir. Conclusion: This study demonstrates that recent clinical isolates of influenza A H3N2 virus no longer agglutinate chicken RBCs, but despite significant receptor binding changes as a result of changes in HA, there was little variation in sensitivity of the NA to zanamivir.

Attenuating mutations in the influenza virus genome which may increase the safety of vaccine production

Influenza virus epidemics occur on an annual basis and cause severe disease in the very young and old. The vaccine administered to high-risk groups is generated by amplifying reassortant viruses, with chronologically relevant viral surface antigens, in eggs. Every 20 years or so, influenza pandemics occur causing widespread fatality in all age groups. These viruses display novel viral surface antigens acquired from a zoonotic source, and vaccination against them poses new issues since production of large amounts of a respiratory virus containing novel surface antigens could be dangerous for those involved in manufacture. To minimise risks, it is advisable to use a virus whose genetic backbone is highly attenuated in man. Traditionally, the A/PR/8/34 strain of virus is used, however, the genetic basis of its attenuation is unclear. Cold-adapted (CA) strains of the influenza virus are all based on the H2N2 subtype, itself a virus with pandemic potential, and again the genetic basis of temperature sensitivity is not yet established. Reverse genetics technology allows us to engineer designer influenza viruses to order. Using this technology, we have been investigating mutations in several different gene segments to effectively attenuate potential vaccine strains allowing the safe production of vaccine to protect against the next pandemic. (C) 2003 Elsevier B.V. All rights reserved.

Cost-benefit evaluation of routine influenza immunisation in people 65-74 years of age

OBJECTIVES: To determine the cost-effectiveness of influenza vaccination in people aged 65-74 years in the absence of co-morbidity. DESIGN: Primary research: randomised controlled trial. SETTING: Primary care. PARTICIPANTS: People without risk factors for influenza or contraindications to vaccination were identified from 20 general practitioner (GP) practices in Liverpool in September 1999 and invited to participate in the study. There were 5875/9727 (60.4%) people aged 65-74 years identified as potentially eligible and, of these, 729 (12%) were randomised. INTERVENTION: Participants were randomised to receive either influenza vaccine or placebo (ratio 3:1), with all individuals receiving pneumococcal vaccine unless administered in the previous 10 years. Of the 729 people randomised, 552 received vaccine and 177 received placebo; 726 individuals were administered pneumococcal vaccine. MAIN OUTCOME MEASURES AND METHODOLOGY OF ECONOMIC EVALUATION: GP attendance with influenza-like illness (ILI) or pneumonia (primary outcome measure); or any respiratory symptoms; hospitalisation with a respiratory illness; death; participant self-reported ILI; quality of life (QoL) measures at 2, 4 and 6 months post-study vaccination; adverse reactions 3 days after vaccination. A cost-effectiveness analysis was undertaken to identify the incremental cost associated with the avoidance of episodes of influenza in the vaccination population and an impact model was used to extrapolate the cost-effectiveness results obtained from the trial to assess their generalisability throughout the NHS. RESULTS: In England and Wales, weekly consultations for influenza and ILI remained at baseline levels (less than 50 per 100,000 population) until week 50/1999 and then increased rapidly, peaking during week 2/2000 with a rate of 231/100,000. This rate fell within the range of 'higher than expected seasonal activity' of 200-400/100,000. Rates then quickly declined, returning to baseline levels by week 5/2000. The predominant circulating strain during this period was influenza A (H3N2). Five (0.9%) people in the vaccine group were diagnosed by their GP with an ILI compared to two (1.1%) in the placebo group [relative risk (RR), 0.8; 95% confidence interval (CI) = 0.16 to 4.1]. No participants were diagnosed with pneumonia by their GP and there were no hospitalisations for respiratory illness in either group. Significantly fewer vaccinated individuals self-reported a single ILI (4.6% vs 8.9%, RR, 0.51; 95% CI for RR, 0.28 to 0.96). There was no significant difference in any of the QoL measurements over time between the two groups. Reported systemic side-effects showed no significant differences between groups. Local side-effects occurred with a significantly increased incidence in the vaccine group (11.3% vs 5.1%, p = 0.02). Each GP consultation avoided by vaccination was estimated from trial data to generate a net NHS cost of 174 pounds. CONCLUSIONS: No difference was seen between groups for the primary outcome measure, although the trial was underpowered to demonstrate a true difference. Vaccination had no significant effect on any of the QoL measures used, although vaccinated individuals were less likely to self-report ILI. The analysis did not suggest that influenza vaccination in healthy people aged 65-74 years would lead to lower NHS costs. Future research should look at ways to maximise vaccine uptake in people at greatest risk from influenza and also the level of vaccine protection afforded to people from different age and socio-economic populations.

Alterations in receptor binding properties of recent human influenza H3N2 viruses are associated with reduced natural killer cell lysis of infected cells

Natural killer (NK) cell recognition of influenza virus-infected cells involves hemagglutinin (HA) binding to sialic acid (SA) on activating NK receptors. SA also acts as a receptor for the binding of influenza virus to its target host cells. The SA binding properties of H3N2 influenza viruses have been observed to change during circulation in humans: recent isolates are unable to agglutinate chicken red blood cells and show reduced affinity for synthetic glycopolymers representing SA-alpha-2,3-lactose (3'SL-PAA) and SA-alpha-2,6-N-acetyl lactosamine (6'SLN-PAA) carbohydrates. Here, NK lysis of cells infected with human H3N2 influenza viruses isolated between 1969 and 2003 was analyzed. Cells infected with recent isolates (1999 to 2003) were found to be lysed less effectively than cells infected with older isolates (1969 to 1996). This change occurred concurrently with the acquisition of two new potential glycosylation site motifs in RA. Deletion of the potential glycosylation site motif at 133 to 135 in HA1 from a recent isolate partially restored the agglutination phenotype to a recombinant virus, indicating that the HA-SA interaction is inhibited by the glycosylation modification. Deletion of either of the recently acquired potential glycosylation sites from HA led to increased NK lysis of cells infected with recombinant viruses carrying modified HA. These results indicate that alterations in RA glycosylation may affect NK cell recognition of influenza virus-infected cells in addition to virus binding to host cells.

Probing the receptor interactions of an H5 avian influenza virus using a baculovirus expression system and functionalised poly(acrylic acid) ligands

Influenza viruses attach to host cells by binding to terminal sialic acid (Neu5Ac) on glycoproteins or glycolipids. Both the linkage of Neu5Ac and the identity of other carbohydrates within the oligosaccharide are thought to play roles in restricting the host range of the virus. In this study, the receptor specificity of an H5 avian influenza virus haemagglutinin protein that has recently infected man (influenza strain A/Vietnam/1194/04) has been probed using carbohydrate functionalised poly(acrylic acid) polymers. A baculovirus expression system that allows facile and safe analysis of the Neu5Ac binding specificity of mutants of H5 HA engineered at sites that are predicted to effect a switch in host range has also been developed. (C) 2007 Elsevier Ltd. All rights reserved.

Is influenza vaccination cost effective for healthy people between ages 65 and 74 years? A randomised controlled trial

The aim of this study was to determine the cost effectiveness of influenza vaccination for healthy people aged 65-74 years living in the UK. People without risk factors for influenza (chronic heart, lung or renal disease, diabetic, immuno-suppressed or those living in an institution) were identified from 20 general practitioner (GP) practices in Liverpool in September 1999. 729/5875 (12.4%) eligible individuals were recruited and randomised to receive either influenza vaccine or placebo (ratio 3: 1)! with all participants receiving 23-valent-pneumococcal polysaccharide vaccine unless already administered. The primary analysis was the frequency of influenza as recorded by a GP diagnosis of pneumonia or influenza like illness. In 2000, the UK vaccination policy was changed with influenza vaccine becoming available. for all people aged 65 years and over irrespective of risk. As a consequence of this policy change. the study had to be fundamentally restructured and only results obtained over a one rather than the originally planned two-year randomised controlled trial framework were used. Results from 1999/2000 demonstrated no significant difference between groups for the primary outcome (relative risk 0.8, 95%, CI 0.16-4.1). In addition. there were no deaths or hospitalisations for influenza associated respiratory illness in either group. The subsequent analysis. using both national and local sources of evidence, estimated the following cost effectiveness indicators: (1) incremental NHS cost per GP consultation avoided = pound2000; (2) incremental NHS cost per hospital admission avoided = pound61,000: (3) incremental NHS cost per death avoided = pound1.900.000 and (4) incremental NHS cost per QALY gained = pound304,000. The analysis suggested that influenza vaccination in this Population would not be cost effective. (C) 2004 Elsevier Ltd. All rights reserved.

Otimização da RT-PCR Multiplex para detecção de vírus dengue em amostras de Aedes aegypti infectados artificialmente

A dengue é a mais importante doença viral transmitida por mosquitos, no que diz respeito à morbidade e mortalidade, que afeta os seres humanos. Este vírus é transmitido pelos vetores Aedes albopictus e Aedes aegypti, este último é o principal vetor nas Américas. O controle da doença se baseia na vigilância laboratorial e vigilância entomológica. A vigilância laboratorial visa aprimorar a capacidade do diagnóstico, detectando precocemente a circulação viral e monitorando os sorotipos circulantes. Dentro deste tipo de vigilância, a RT-PCR é um método bastante usado no diagnóstico da doença em humanos e mosquitos, porém, a má conservação do material pode comprometer a integridade do RNA e trazer resultados falso-negativos. O desenvolvimento de melhores métodos de vigilância do vírus dengue (DENV) em mosquitos é de grande valor para os programas de controle. Desta maneira, o presente projeto visou otimizar a técnica de RT-PCR Multiplex para detecção de DENV em amostras de Ae. aegypti infectadas artificialmente pelo vírus. Primers que amplificam uma região de 80 pb do gene rpL8 de mosquito foram desenhados no site Primer3 e avaliados na ferramenta online Multiple Primer Analyzer, junto com primers que amplificam os sorotipos DENV. Não houve competição de primers e foi observado bandas distintas no gel de agarose. Foi avaliado o efeito de diferentes formas de preservação do material genético das amostras (RNAlater®, freezer -80°C e nitrogênio líquido) por 7 dias, onde não houve diferenças significativas em relação à integridade do RNA. O efeito de diferentes formas de extração de RNA (Kit da QIAGEN® , TRIzol® e Chomczymski-Sacchi) também foi avaliado e o método ChomczymskiSacchi obteve o melhor desempenho. A otimização desta técnica permitirá uma maior confiabilidade nos resultados, já que além da detecção dos sorotipos, haverá uma confirmação da qualidade do RNA, aprimorando a capacidade do diagnóstico e auxiliando a prevenção e controle da transmissão da dengue.