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Monoclonal antibodies for the rapid diagnosis of influenza A and B virus infections by immunofluorescence.

McQuillin J, Madeley CR, Kendal AP.

Mouse monoclonal antibodies, directed against antigenic sites on influenza A and B viruses and found to be type-specific in an immunoassay, were assessed for use as diagnostic reagents in an indirect immunofluorescence assay on nasopharyngeal secretions. The influenza A antibodies were directed against nucleoprotein or matrix protein antigens and the influenza B antibodies against nucleoprotein and haemagglutinin antigens. The influenza A anti-matrix monoclonal antibody was found to give a strong intranuclear particulate fluorescence in normal baboon kidney cells and cells from nasopharyngeal secretions negative for influenza A virus, including those from a patient infected with respiratory syncytial virus. Pools of the remaining monoclonal antibodies gave satisfactory results on 25 specimens from patients with influenza A H1N1 and H3N2 subtypes and 12 from patients with influenza B.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2865418&dopt=Abstract flu, influenza



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Influenza, pneumococcal, and tetanus toxioid vaccination of adults--United States, 1993-7.

Singleton JA, Greby SM, Wooten KG, Walker FJ, Strikas R.

Epidemiology and Surveillance Division, National Immunization Program, USA.

PROBLEM/CONDITION: An increasing proportion of adults have received recommended vaccinations against influenza, pneumococcal infection, and tetanus. However, in 1995, fewer than 60% of adults were vaccinated as recommended. REPORTING PERIOD COVERED: 1993-1997. DESCRIPTION OF SYSTEM: Data were obtained from the state-based Behavioral Risk Factor Surveillance System (BRFSS) for 1993, 1995, and 1997 and from the National Health Interview Survey (NHIS) for 1995 to describe national, regional, and state-specific patterns of use of influenza and pneumococcal vaccines and tetanus toxoid among noninstitutionalized adults aged > or = 18 years. RESULTS: Among adults aged > or = 65 years in 1995, 58% reported receiving an influenza vaccination during the previous 12 months, and 34% reported ever receiving a pneumococcal vaccination. In this age group, non-Hispanic whites were more likely to report receipt of influenza (61%) and pneumococcal vaccines (36%) than non-Hispanic blacks (40% and 22%, respectively) and Hispanics (50% and 23%, respectively). Among the 50 states and the District of Columbia, the median vaccination level among older adults (i.e., persons aged > or = 65 years) increased from 51% in 1993 to 66% in 1997 for influenza vaccine, and from 28% in 1993 to 46% in 1997 for pneumococcal vaccine. Adults with chronic medical conditions had low vaccination levels. Those aged 50-64 years were more likely than those aged 18-49 years to report influenza (38% versus 20%) and pneumococcal vaccination (20% versus 12%). In 1995, the proportion of adults who reported receiving a tetanus vaccination during the previous 10 years decreased with age, from 65% among those aged 18-49 years to 54% among those aged 50-64 years and to 40% among those aged > or = 65 years. In each age group, women were less likely than men to report receiving tetanus toxoid; and among adults aged > or = 65 years, Hispanics and Asians/Pacific Islanders were least likely among all racial/ethnic groups to report receiving tetanus toxoid. INTERPRETATION: By 1995, the Healthy People 2000 objective to increase to at least 60% the proportion of persons aged > or = 65 years who had received annual influenza vaccination had been achieved among non-Hispanic whites (objective 20.11). However, substantial improvement is needed among non-Hispanic blacks, Hispanics, and adults aged < 65 years with high-risk medical conditions. PUBLIC HEALTH ACTIONS: Continued surveillance of vaccine coverage among adults will direct attention to undervaccinated populations that may be disproportionately affected by vaccine-preventable diseases. Vaccination coverage data can be used to guide efforts to increase awareness among health-care providers and the public about the benefits of vaccination, establish systems to ensure that every contact with the health-care system is used to update vaccinations, and further support financial mechanisms to increase vaccine delivery.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11016877&dopt=Abstract flu, influenza



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Enzymological heterogeneity of influenza B virus neuraminidase demonstrated by the fluorometric assay method.

Kiyotani K, Takei N, Matsuo Y.

The neuraminidase activity of 26 strains of influenza B virus isolated from all over the world was investigated colorimetrically, using fetuin as a substrate, and fluorometrically, using 4-methylumbelliferyl(4-MU)-N-Ac-alpha-D-neuraminide as a substrate, with special reference to enzymological heterogeneity. The activity of influenza A viral neuraminidases and of a commercially available pure viral one was strongly inactivated by either ethylenediaminetetraacetic acid or glycoletherdiaminetetraacetic acid, when measured by the fluorometric assay method, whereas that of influenza B ones was not at all. However, the viral neuraminidases of both influenza A and B viruses were found to be calcium ion-dependent by the colorimetric assay method. A difference in the catalytic rate between the two assay methods was observed with influenza B viral neuraminidase to a much greater extent as compared with that of influenza A. A difference in substrate specificity of these enzymes was demonstrated to be due to a difference in the degree of competitive inhibition by N-acetylneuraminic acid. These findings strongly suggest that enzymological heterogeneity in influenza B viral neuraminidase may be attributed to delicate structural differences, between the enzymes of influenza A and B viruses demonstratable only by the fluorometric neuraminidase assay method using 4-MU-N-Ac-alpha-D-neuraminide as a substrate.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2867656&dopt=Abstract flu, influenza



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Expression of influenza virus NS2 nonstructural protein in bacteria and localization of NS2 in infected eucaryotic cells.

Greenspan D, Krystal M, Nakada S, Arnheiter H, Lyles DS, Palese P.

The nonstructural NS2 protein of influenza A/PR/8/34 virus was efficiently expressed in bacteria, and monospecific antisera were prepared against the bacterially synthesized polypeptide. These antisera were cross-reactive among the NS2 proteins of various influenza A viruses. However, they did not react with the NS2 of influenza B/Lee/40 virus nor with other proteins of influenza A viruses such as NS1. Antisera against NS2 were used to determine that the NS2 protein is localized in the cell nucleus during influenza virus infection, as shown by immunofluorescence microscopy. Cells infected with simian virus 40 recombinants containing the influenza virus NS gene revealed that both the NS1 and NS2 proteins appeared in the nucleus, even in the absence of expression of other influenza virus-specific components.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2987535&dopt=Abstract flu, influenza



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Immunoglobulin G subclass antibody responses in influenza A and parainfluenza type 1 virus infections.

Julkunen I, Hovi T, Seppala I, Makela O.

Antibody responses in immunoglobulin G1, G2, G3, G4, A (IgA1) and M isotypes were studied in 10 patients with an acute influenza A and in another 10 patients with a parainfluenza type 1 virus infection using radioimmunoassay with standardized monoclonal anti-immunoglobulins. A four-fold or greater increase of antibody in patients have an acute influenza A virus infection, were found in IgG1 (all 10 cases), IgG3 (seven cases), IgG4 (eight cases) and in IgA1 (six cases) whereas IgG2 and IgM responses were observed only in one and three cases, respectively. The antibody titre values were converted to immunoglobulin units by multiplying the titre by a pre-determined correction coefficient compensating for the varying affinity of the individual monoclonal anti-immunoglobulins. These units were then used to calculate the actual proportions of each isotype. In the convalescent phase, 78% of total anti-influenza A antibodies were estimated to be of IgG1 isotype and other immunoglobulin isotypes varied from 3 to 7% of total. Similar results in parainfluenza virus antibodies were obtained with serum pairs from patients with an acute parainfluenza virus infection.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2988830&dopt=Abstract flu, influenza



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[Incidence of amantadine-resistant influenza A (genotype Ser-31-Asn) in nursing homes in Niigata, Japan]

[Article in Japanese]

Saito R, Masuda H, Oshitani H, Suzuki H, Kawasaki S, Sato H.

Department of Public Health, Niigata University, School of Medicine.

The antiviral agent amantadine specifically inhibit influenza A virus infection, but the emergence of drug-resistant viruses occur more readily with amantadine treatment. In human influenza viruses, single amino acid changes at 4 sites spanning the transmembrane domain of the M2 protein can confer drug resistance. Amantadine was approved for treatment of Parkinson's disease in 1975, and for the influenza A virus infection in November 1998, in Japan. Annual consumption of amantadine for influenza treatment increased suddenly after the approval. According to our previous report, the predominant genotype of resistant virus is the substitution S-31-N in M2 both in vitro and in clinical samples, as in the other reports. Based on the above findings, we focused on single amino acid change at position 31 (genotype S-31-N) and applied polymerase chain-reaction restriction fragment length polymorphism (PCR-RFLP), directly from throat swab samples, by using primers that insert a restriction site for Sca I. With this technique, we surveyed the incidence of amantadine resistant viruses in nursing homes, Niigata, Japan. Thirty-one (22.0%) of 141 PCR positive samples from 8 nursing homes in 1998-99 season showed resistant patterns, and only 6 (19.4%) of them were after the administration of amantadine for treatment. All of these 8 nursing homes used amantadine for Parkinson's disease, but only half of them used the drug for influenza A infection. The incidence of resistant viruses was not significantly different from facilities with amantadine for influenza treatment to those without, 25.5% and 14.0% respectively. The occurrence of outbreaks and sporadic illness in those facilities, with different administration status were observed, but fortunately we could not find any evidence to relate the emergence of resistant viruses to the outbreaks. This is the first report that the resistant influenza viruses already exist in nursing facilities where amantadine was used for not only influenza but also Parkinson's disease in Japan.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11019512&dopt=Abstract flu, influenza



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Noncumulative sequence changes in the hemagglutinin genes of influenza C virus isolates.

Buonagurio DA, Nakada S, Desselberger U, Krystal M, Palese P.

Sequence analysis and comparison of hemagglutinin (HA) genes of different influenza C viruses isolated between 1947 and 1983 reveals that (1) the extent of difference among the HA genes is independent of the year in which these viruses were isolated and that (2) changes in the HA genes do not appear to accumulate with time. These results suggest that epidemiologically dominant variants of influenza C viruses do not emerge successively with time and that C virus variants derived from multiple evolutionary pathways cocirculate at any one time. Thus the epidemiology of influenza C viruses differs markedly from that of influenza A viruses, which is characterized by the emergence of successive variants. Based on the nucleotide sequence data, we propose different evolutionary models for influenza A and influenza C viruses.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3855244&dopt=Abstract flu, influenza



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Influenza A virus nucleoprotein is a major target antigen for cross-reactive anti-influenza A virus cytotoxic T lymphocytes.

Yewdell JW, Bennink JR, Smith GL, Moss B.

Influenza A virus-specific cytotoxic T lymphocytes (CTL) capable of lysing cells infected with any influenza A virus ("cross-reactive CTL") constitute a major portion of the host CTL response to influenza. The viral nucleoprotein (NP), a major internal virion structural protein, has been implicated as a possible target antigen for cross-reactive CTL. To directly examine CTL recognition of NP, a vaccinia virus recombinant containing a DNA copy of an influenza A virus NP gene was constructed. We found that murine cells infected with this virus were efficiently lysed in a major histocompatibility complex-restricted manner by cross-reactive CTL populations obtained by immunization with a variety of influenza A virus subtypes. In addition, the recombinant vaccinia virus containing the PR8 NP gene was able to both stimulate and prime for a vigorous secondary cross-reactive CTL response. Significantly, splenocytes from mice primed by inoculation with the recombinant vaccinia virus containing the PR8 NP gene could be stimulated by influenza A viruses of all three major human subtypes. Finally, unlabeled target competition experiments suggest that NP is a major, but not the sole, viral target antigen recognized by cross-reactive CTL.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3872457&dopt=Abstract flu, influenza



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Human peripheral blood mononuclear cells produce IgA anti-influenza virus antibody in a secondary in vitro antibody response.

Yarchoan R, Barrow LA, Kurman C, Strober W, Nelson DL.

The function and immunoregulation of human IgA memory B cells producing anti-influenza virus antibody was analyzed in vitro in antigen-stimulated cultures. Peripheral blood mononuclear cells (PBMC) from seven of eight normal adult volunteers naturally immunized to influenza virus produced IgA anti-influenza virus antibody when stimulated in vitro with inactivated A/Aichi/68 [H3N2] influenza virus. This IgA antibody response was approximately one-eighth the IgG antibody response. PBMC from each of five patients with selective IgA deficiency failed to produce any measurable IgA antibody. When tonsillar mononuclear cells (TMC) were studied in a similar manner, a relatively higher IgA antibody response was obtained (one-third the IgG antibody) than with PBMC. Additional studies were undertaken to investigate the immunoregulation of this IgA antibody production and the relatively lower amount produced by PBMC than by TMC. Co-cultures of peripheral blood B cells with irradiated peripheral blood T cells (to possibly inactivate a radiosensitive IgA suppressor cell) did not result in a relative increase in IgA antibody production. Also, co-cultures of B cells with increasing numbers of T cells produced parallel increases of IgG and IgA antibody when plotted on a log scale with slopes of approximately 1, suggesting that a single helper T cell was limiting for both isotypes. Finally, pokeweed mitogen-stimulated co-cultures of peripheral blood and tonsillar B and T cells revealed that the B cell population, but not the T cell population, determined the amount of IgA anti-influenza virus antibody produced. Precursor frequency analyses of tonsillar and peripheral blood B cells in antigen-stimulated cultures confirmed that tonsils contained a higher precursor frequency of B cells for IgA anti-influenza virus antibody production (3.95/10(6) B cells) than did peripheral blood B cells (0.65/10(6) B cells). Thus, IgA memory cells are preferentially found in tonsillar tissue as compared with the peripheral blood, consistent with the role of the tonsils as a mucosal immune organ.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3874224&dopt=Abstract flu, influenza



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[Use of a method of molecular nucleic acid hybridization for the rapid diagnosis of influenza]

[Article in Russian]

Baurin VV, Tentsov IuIu, Ivanova LA, Bukrinskaia AG.

A highly sensitive method of pinpoint hybridization of nucleic acids on nitrocellulose filters using 32P-labeled pHA plasmid carrying a DNA copy of hemagglutinin gene of influenza A/Udorn/307/72 (H3N2) was developed which permitted specific detection of minimal amounts of RNA (units of pikograms) of influenza A virus with H3 serotype hemagglutinin. The method of pinpoint hybridization was used for the detection of RNA of influenza A (H3 serotype) in nasopharyngeal washings of patients with acute respiratory diseases during the influenza outbreak of February-March, 1984. In parallel, the presence of viral antigen was determined by direct immunofluorescence using H3N2 antiserum, and the diagnosis of influenza was confirmed by the clinical picture of the disease. The results indicate that the pinpoint hybridization method may be used for rapid diagnosis of influenza as a highly sensitive and specific tool.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3907140&dopt=Abstract flu, influenza



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Incidence and clinical manifestations of influenza in nurse assistant students.

Sirivichayakul C, Sabcharoen A, Chanthavanich P, Chokejindachai W, Thawatsupha P, Suthisarnsunthorn U, Ounkaew W.

Department of Tropical Pediatrics, Faculty of Tropical Medicine, Mahidol University, Thailand.

A prospective study was conducted to find the incidence and clinical manifestations of influenza in 201 nurse assistant students of Faculty of Tropical Medicine during June 1998 to May 1999. There were 106 episodes of influenza-like illness (incidence 52.7%) of which only 33% were proven to be influenza (incidence 17.4%). Main clinical manifestations of influenza included headache, fever, malaise, myalgia, rhinorrhea, cough, and sore throat. We found that influenza could not be diagnosed solely by using clinical manifestations. Respiratory pathogenic bacteria were rarely isolated in patients with influenza-like illness and this led to our suggestion that routine pharyngeal culture and antibiotic therapy would not be helpful. Influenza vaccination of every nurse assistant student would be beneficial.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11023065&dopt=Abstract flu, influenza