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In vitro and in vivo inhibitory effects of disodium cromoglycate on influenza virus infection.

Hidari KI, Tsujii E, Hiroi J, Mano E, Miyatake A, Miyamoto D, Suzuki T, Suzuki Y.

Department of Biochemistry, University of Shizuoka, School of Pharmaceutical Sciences, Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Yada, Shizuoka, Japan. hidari u-shizuoka-ken.ac.jp

Disodium cromoglycate (DSCG) is one of the safest drugs for the prevention of bronchial asthma and allergic rhinitis attacks. The effect of DSCG on acute upper respiratory tract viral infection is still controversial. Here we investigated DSCG inhibition of influenza virus infection in vivo and in vitro. In vivo effects of DSCG on viral infection were assessed using a murine model of respiratory tract infection. Intranasal administration of DSCG protected mice from death induced by infection with influenza virus A/PR/8/34. We analyzed DSCG anti-viral effects in vitro by either (i) treating cells prior to viral adsorption, (ii) treating cells concurrently with viral adsorption, or (iii) treating cells after viral adsorption. DSCG treatment of cells during or after, but not before, viral adsorption significantly inhibited influenza viral infection, indicating DSCG acts on events late in viral infection. DSCG exerts anti-influenza effect both in vitro and in vivo at the doses compatible with treatment for asthma. DSCG marginally inhibited influenza viral neuraminidase and membrane fusion functions, suggesting that DSCG inhibition of viral neuraminidase and fusion activities may partially mediate this anti-influenza effect. Our results indicate that treatment of patients including children with DSCG may take advantages for prevention from influenza virus infection.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15187427&dopt=Abstract flu, influenza



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A cohort study of the effectiveness of influenza vaccine in older people, performed using the United Kingdom general practice research database.

Mangtani P, Cumberland P, Hodgson CR, Roberts JA, Cutts FT, Hall AJ.

London School of Hygiene and Tropical Medicine, London, United Kingdom. punam.mangtani lshtm.ac.uk

BACKGROUND: The effectiveness of influenza vaccination against hospitalization and death can only ethically be assessed in observational studies. A concern is that individuals who are vaccinated are healthier than individuals who are not vaccinated, potentially biasing estimates of effectiveness upward. METHODS: We conducted a historical cohort study of individuals >64 years of age, for whom there were data available in the General Practice Research Database for 1989 to 1999 in England and Wales. Rates of admissions for acute respiratory diseases and rates of death due to respiratory disease were compared over 692,819 person-years in vaccine recipients and 1,534,280 person-years in vaccine nonrecipients. RESULTS: The pooled effectiveness of vaccine against hospitalizations for acute respiratory disease was 21% (95% confidence interval [CI], 17%-26%). The rate reduction attributable to vaccination was 4.15 hospitalizations/100,000 person-weeks in the influenza season. Among vaccine recipients, no important reduction in the number of admissions to the hospital was seen outside influenza seasons. The pooled effectiveness of vaccine against deaths due to respiratory disease was 12% (95% CI, 8%-16%). A greater proportionate reduction was seen among people without medical disorders, but absolute rate reduction was higher in individuals with medical disorders, compared with individuals without such disorders (6.14 deaths due to respiratory disease/100,000 person-weeks vs. 3.12 deaths due to respiratory disease/100,000 person-weeks). Clear protection against death due to all causes was not seen. CONCLUSIONS: Influenza vaccination reduces the number of hospitalizations and deaths due to respiratory disease, after correction for confounding in individuals >64 years of age who had a high risk or a low risk for influenza. For elderly people, untargeted influenza vaccination is of confirmed benefit against serious outcomes.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15195237&dopt=Abstract flu, influenza



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[Influenza in Poland in 2002]

[Article in Polish]

Kuszewski K, Brydak LB, Machala M.

Zaklad Epidemiologii Panstwowego Zakladu Higieny ul. Chocimska 24, 00-791 Warszawa. kkuszewski pzh.gov.pl

In 2002 the number of cases of influenza and influenza-like illness (ILI) registered in Poland amounted to 228,055. This is 39.5% of the number of cases recorded in 2001. The highest influenza incidence was found in Mazowieckie voivodship (2297.5 per 100,000), while the lowest incidence was registered in Swietokrzyskie voivodship (104.1) and Lubelskie voivodship (117.6). In children aged 0 to 14 years the number of influenza and ILI cases amounted to 104,552 (incidence of 1511.9 per 100,000) and this is 46% of the total number of cases recorded in 2002. The number of patients referred to hospitals amounted to 223, including 92 children aged 0-14 years. There were isolated 10 influenza strains, including 7 strains of subtype A (H3N2) and 3 strains of subtype A (H1N1). Immunofluorescence test carried out in 57 specimens did not confirm infection with influenza virus. Sero-surveys showed the lowest antihemagglutinin antibody levels in the age groups 0-3 and 4-7 years, while the highest in people aged 8-14 and 15-25 years.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15218642&dopt=Abstract flu, influenza



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Immunostimulant patch enhances immune responses to influenza virus vaccine in aged mice.

Guebre-Xabier M, Hammond SA, Ellingsworth LR, Glenn GM.

IOMAI Corporation, Gaithersburg, MD 20878, USA.

Improvement in the immune response to influenza virus vaccination in the elderly represents the primary unmet need in influenza virus vaccination. We have shown that topical application of immunostimulating (IS) patches containing heat-labile enterotoxin of Escherichia coli (LT) enhances immune responses to injected vaccines. We extend these findings and show that LT-IS patch application enhances the antibody responses to influenza virus vaccination in both young and aged mice. LT-IS patches markedly increased influenza virus-specific immunoglobulin G (IgG), hemagglutination inhibition antibody, mucosal antibody, and T-cell responses. The magnitude of the immune responses in aged mice receiving an LT-IS patch was equivalent to or greater than that of the immune responses in young mice given vaccine alone. These results suggest that addition of an LT-IS patch may compensate for the deficient immune function seen in the aged in response to influenza virus vaccination. Therefore, use of an LT-IS patch could be a new, safe, and simple immunization strategy that may significantly improve the outcome of influenza virus vaccination in the elderly.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15220436&dopt=Abstract flu, influenza



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Influenza-like viral illnesses and flare-ups of fibrodysplasia ossificans progressiva.

Scarlett RF, Rocke DM, Kantanie S, Patel JB, Shore EM, Kaplan FS.

Department of Orthopaedic Surgery, The University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

Flare-ups of fibrodysplasia ossificans progressiva are most commonly triggered by soft tissue trauma. After observing severe flare-ups of fibrodysplasia ossificans progressiva in two half-sisters with culture-confirmed influenza B infections, we hypothesized that influenza-like viral illnesses also can trigger fibrodysplasia ossificans progressiva flare-ups. To address this hypothesis, we designed a questionnaire to assess whether patients with fibrodysplasia ossificans progressiva experienced influenza symptoms during the 2000 to 2001 influenza season, and whether these symptoms were correlated with flare-ups of the condition. The questionnaire was sent to patients with fibrodysplasia ossificans progressiva worldwide. Of the 264 patients surveyed, 123 (47%) responded. The survey revealed that the risk of a disease flare-up of fibrodysplasia ossificans progressiva during an influenza-like viral illness was increased at least threefold and possibly much more. The survey data strongly supported the hypothesis that influenza-like viral illnesses are associated with disease flare-ups in patients who have fibrodysplasia ossificans progressiva. Influenza-like viral illnesses may be a source of previously unrecognized muscle injury leading to heterotopic ossification and permanent loss of mobility in these patients. These findings have important implications for understanding and preventing environmental triggers of disease activity in this population of patients genetically susceptible to progressive heterotopic ossification.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15232462&dopt=Abstract flu, influenza



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Comparative study of influenza virus replication in Vero and MDCK cell lines.

Youil R, Su Q, Toner TJ, Szymkowiak C, Kwan WS, Rubin B, Petrukhin L, Kiseleva I, Shaw AR, DiStefano D.

Merck & Co., Inc. 770 Sumneytown Pike, WP44L-206B, West Point, PA 19486, USA. rima_youil merck.com

The choice of a cell line for the production of influenza vaccines is determined by how well the virus is able to replicate and how easily the cell line can be maintained. Madin-Darby canine kidney (MDCK) cells have long been known to successfully support influenza growth. Vero cells are also another well studied candidate cell line. In this work, we have compared these two cell lines for their ability to propagate type A and type B cold-adapted and wild type influenza viruses. The growth of these viruses has been measured as plaque forming units (via plaque assay) as well as viral particle formation (via a novel quantitative RT-PCR assay) to assess the suitability of these cell lines to support the development of live attenuated influenza vaccines. The novel qRT-PCR assay outlined in this work was demonstrated to be an efficient, sensitive and reproducible method for measuring wild type (wt) and cold-adapted (ca) influenza strains. Replicates of six per sample consistently showed an average variation around +/-10%. In this study we have also found qRT-PCR to be a useful method for differentiating between wt and ca influenza strains based on their differing growth characteristics at varying temperatures. This can subsequently be used to assess reassortants prepared from ca donor strains for the purposes of live viral vaccine development. For type A and B influenza viruses studied in this work, MDCK cells supported a more rapid viral growth (measured in terms of genome copies) compared with Vero cells. For the type A viruses studied here, the genome copies: infectious unit (genome copy, gc:infectious unit, iu) ratio was found to be more favorable for Vero cells compared with MDCK cells. For the type B viruses studied in this work, the gc:iu was equivalent in both cell lines tested. Ultimately, however, the use of any new cell line would need to be approved by regulatory agencies prior to its commercial application.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15234806&dopt=Abstract flu, influenza



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The evolution of H5N1 influenza viruses in ducks in southern China.

Chen H, Deng G, Li Z, Tian G, Li Y, Jiao P, Zhang L, Liu Z, Webster RG, Yu K.

Animal Influenza Laboratory of the Ministry of Agriculture, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Harbin 150001, People's Republic of China. hlchen1 yahoo.com

The pathogenicity of avian H5N1 influenza viruses to mammals has been evolving since the mid-1980s. Here, we demonstrate that H5N1 influenza viruses, isolated from apparently healthy domestic ducks in mainland China from 1999 through 2002, were becoming progressively more pathogenic for mammals, and we present a hypothesis explaining the mechanism of this evolutionary direction. Twenty-one viruses isolated from apparently healthy ducks in southern China from 1999 through 2002 were confirmed to be H5N1 subtype influenza A viruses. These isolates are antigenically similar to A/Goose/Guangdong/1/96 (H5N1) virus, which was the source of the 1997 Hong Kong "bird flu" hemagglutinin gene, and all are highly pathogenic in chickens. The viruses form four pathotypes on the basis of their replication and lethality in mice. There is a clear temporal pattern in the progressively increasing pathogenicity of these isolates in the mammalian model. Five of six H5N1 isolates tested replicated in inoculated ducks and were shed from trachea or cloaca, but none caused disease signs or death. Phylogenetic analysis of the full genome indicated that most of the viruses are reassortants containing the A/Goose/Guangdong/1/96-like hemagglutinin gene and the other genes from unknown Eurasian avian influenza viruses. This study is a characterization of the H5N1 avian influenza viruses recently circulating in ducks in mainland China. Our findings suggest that immediate action is needed to prevent the transmission of highly pathogenic avian influenza viruses from the apparently healthy ducks into chickens or mammalian hosts.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15235128&dopt=Abstract flu, influenza



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Lung-specific expression of active Raf kinase results in increased mortality of influenza A virus-infected mice.

Olschlager V, Pleschka S, Fischer T, Rziha HJ, Wurzer W, Stitz L, Rapp UR, Ludwig S, Planz O.

Institut fur Immunologie, Bundesforschungsanstalt fur Viruskrankheiten der Tiere, Paul-Ehrlich-Str 28, Tubingen, Germany.

Alterations in signalling via the Raf/MEK/ERK pathway interfere with influenza A virus replication in cell culture. While virus yields are reduced in cells expressing dominant-negative Raf or ERK, virus propagation is enhanced upon expression of constitutively active Raf or MEK. To study the impact of active Raf on influenza virus propagation in vivo, we investigated transgenic mice expressing an activated mutant of c-Raf (Raf-BxB) in the main target tissue of influenza virus, the lung. Raf-BxB expression results in multicentric alveolar adenomas. Influenza virus A infection of Raf-BxB mice results in increased disease symptoms and higher mortality rates. The immune response against viral pathogens in transgenic animals did not differ from wild-type mice as determined by the use of a Pseudorabies virus (PRV) as a model for a viral infection not affecting the lung. No significant differences of influenza virus titers in the lung of Raf-BxB and wild-type mice were observed. However, immunohistology revealed increased numbers of influenza NP-positive cells in the alveolar linings of Raf-BxB mice, demonstrating the strong tropism of influenza virus for cells expressing active Raf. These findings disclose the possibility to use modified influenza virus for the therapy of tumors with an activated Ras/Raf signalling pathway.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15235583&dopt=Abstract flu, influenza



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Chemokine expression during the development and resolution of a pulmonary leukocyte response to influenza A virus infection in mice.

Wareing MD, Lyon AB, Lu B, Gerard C, Sarawar SR.

La Jolla Institute for Allergy and Immunology, San Diego, CA, USA.

Influenza A virus replicates in the respiratory epithelium and induces an inflammatory infiltrate comprised of mononuclear cells and neutrophils. To understand the development of the cell-mediated immune response to influenza and how leukocyte trafficking to sites of inflammation is regulated, we examined the chemokine expression pattern in lung tissue from A/PR/8/34-infected C57BL/6 mice using an RNase protection assay. Monocyte chemoattractant protein 1, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, MIP-3alpha, regulated on activation, normal T expressed and secreted (RANTES), MIP-2, and interferon-inducible protein 10 (IP-10) mRNA expression was up-regulated between days 5 and 15 after infection, consistent with a role for these chemokines in leukocyte recruitment to the lung. Low levels of expression were detected for the CC chemokine receptors (CCR)2 and CCR5, whereas CXC chemokine receptor (CXCR)3 was significantly up-regulated by day 10 after infection, coinciding with peak inflammatory cell infiltration in the airways. As RANTES, IP-10, and their receptors were up-regulated during influenza virus infection, we investigated leukocyte recruitment and viral clearance in mice deficient in RANTES or CXCR3, the receptor for IP-10. Leukocyte recruitment and viral replication in influenza-infected RANTES knockout(-/-) mice were similar to that in control mice, showing that RANTES is not essential for the immune response to influenza infection. Similarly, leukocyte recruitment and viral replication in CXCR3-/- mice were identical to control mice, except at day 8 postinfection, where fewer lymphocytes, neutrophils, and eosinophils were detected in the bronchoalveolar lavage of CXCR3-/- mice. These studies suggest that although the chemokines detected may play a role in regulating leukocyte trafficking to the lung during influenza infection, some may be functionally redundant.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15240757&dopt=Abstract flu, influenza



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Mucosal immunization with attenuated Shigella flexneri harboring an influenza hemagglutinin DNA vaccine protects mice against a lethal influenza challenge.

Vecino WH, Quanquin NM, Martinez-Sobrido L, Fernandez-Sesma A, Garcia-Sastre A, Jacobs WR Jr, Fennelly GJ.

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

Mucosal surfaces are important for the induction of immunity against influenza virus. In a murine intranasal immunization model, we demonstrated that the attenuated Shigella flexneri Deltaasd strain 15D, carrying a DNA construct encoding the influenza virus hemagglutinin (HA), induces protective immunity against a lethal respiratory challenge with influenza A/WSN/33. Influenza virus-specific IFN-gamma T cells were detected among splenocytes, and anti-HA IgG was detected in serum post-immunization, albeit at low levels. Following influenza virus challenge, an accelerated anti-HA IgA antibody response was detected in bronchoalveolar lavage (BAL) washings from mice vaccinated with attenuated shigella containing the HA construct. These results suggest that S. flexneri Deltaasd strain 15D is a promising vector for mucosal DNA vaccine immunization against influenza virus and other mucosal pathogens.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15246259&dopt=Abstract flu, influenza



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Genetic diversity of influenza B virus: the frequent reassortment and cocirculation of the genetically distinct reassortant viruses in a community.

Matsuzaki Y, Sugawara K, Takashita E, Muraki Y, Hongo S, Katsushima N, Mizuta K, Nishimura H.

Department of Bacteriology, Yamagata University School of Medicine, Iida-Nishi, Yamagata, Japan. matuzaki med.id.yamagata-u.ac.jp

To characterize the genetic diversity of influenza B viruses isolated during one influenza season, the antigenic and genetic relationships among 20 strains of influenza B virus isolated in February and March 2001 at one pediatric clinic in Yamagata City, Japan, were investigated. The HA gene and seven other gene segments were phylogenetically divided into three distinct sublineages (Harbin/7/94-, Tokyo/6/98-, and Shiga/T30/98-related lineage) of the Yamagata/16/88-like lineage. The NS genes of the viruses belonging to the Harbin/7/94-related lineage have additional three nucleotides at positions 439-447, and were phylogenetically distinguishable from those of the currently circulating Yamagata/16/88- and Victoria/2/87-like lineages, but were closely related to that of the Yamagata/16/88-like lineage isolated before 1994. Moreover, four strains of influenza B virus isolated in the same community between 2002 and 2003 were further examined. Phylogenetic analysis revealed that a virus of Victoria/2/87-like lineage isolated in 2003 had acquired the NA, NS, M, and PA gene segments from a Shiga/T30/98-like virus, and two strains of Harbin/7/94-related lineage had acquired the various gene segments from Shiga/T30/98-like virus through a reassortment event. These results indicate that genetically distinct multiple viruses can combine to cause an influenza B epidemic in a community and that the frequent reassortment among these viruses plays a role in generating the genetic diversity of influenza B viruses.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15258979&dopt=Abstract flu, influenza