Rapid diagnosis of influenza ( flu ) type A infection: comparison of shell-vial culture, directigen flu-A and enzyme-linked immunosorbent assay.

Two direct antigen detection methods (Directigen FLU A, Becton-Dickinson, Cokeysville, Md. and Influenza ( Flu ) A virus ELISA Antigen detection kit, Alpha Biotech, Milano) were compared with isolation by shell-vial cultures for rapid diagnosis of influenza ( flu ) type A virus infection. The three methods were performed using a reference viral suspension consisting of a clinical isolate of influenza ( flu ) type A virus as well as 41 nasopharyngeal aspirates collected from patients with influenza ( flu ) symptoms. Influenza ( Flu ) type A virus was recovered from 14 specimens by shell-vials. Ten specimens were positive by Directigen FLU A and 7 by ELISA antigen detection kit. When performed on clinical samples the sensitivity of Directigen FLU A and ELISA was 64.2% and 42.8%, respectively. The limit of sensitivity of the shell-vials, performed on a virus stock suspension titrated by haemagglutinin activity, was 0.06-0.03 HA units/ml at 24 hours or 72 hours after inoculation. Directigen FLU A and ELISA detected as positive a virus stock suspension containing 0.25 HA units/ml and 1 HA units/ml, respectively.

Influenza ( Flu ) vaccination--knowledge, attitudes, coverage--can they be improved?

The reasons for low rate of influenza ( flu ) vaccination in Slovakia have been analyzed in selected target groups. In our questionnaire study we focused on the level of knowledge about this vaccination and the attitudes towards it. We selected three target groups: medical students, nurses and printing company workers. The authors as well tried to identify the ways how the flu vaccination coverage could be increased in the future. The questionnaire survey revealed several surprising facts. Though almost all the respondents knew about the existence of influenza ( flu ) vaccine, less than one quarter of them have ever received influenza ( flu ) shot. Despite our expectations that the main source of information about influenza ( flu ) prevention in medical students and nurses would be from their medical and nursing studies, it was shown to be from mass media instead. Even more staggering was the distrust towards the vaccination as a reason for not being vaccinated in a high proportion of both the medical students and the nurses. The majority of medical students would not even want to get a vaccination, even if it were to be provided for free. These results suggest that if we want to improve the low influenza ( flu ) vaccination coverage within the general population of our country, we will have to focus our attention primarily on the professional groups of medical workers and medical and nursing students who should be able to provide the public with the competent advice. Therefore, changing the current negative approach and improving the deficit in knowledge concerning vaccination are the key tasks for all under- as well as postgraduate teachers of medicine and nursing in Slovakia, especially, but not exclusively, of those specialised in public health.(Tab. 3, Fig. 2, Ref. 8).

Amantadine susceptibility in influenza ( flu ) A virus isolates: determination methods and lack of resistance in a Canadian sample, 1991-94.

BACKGROUND: Influenza ( Flu ) A infections are an important cause of morbidity and mortality in the elderly and patients affected by chronic diseases or immunodeficiencies. Treatment and prevention of infection in hospitals and nursing homes often involve the use of amantadine, but resistant viruses may arise. OBJECTIVES: To assess the effectiveness of specific and sensitive methods for rapid screening and sequence confirmation of amantadine resistance, and the occurrence of amantadine resistance in recent influenza ( flu ) A virus isolates in Canada. STUDY DESIGN: A chicken antiserum-based enzyme linked immunoassay (ELISA) was developed and used to screen fifty influenza ( flu ) A isolates for amantadine resistance. Drug sensitivity was expressed as a percentage of virus growth inhibition. The efficiency of the assay was compared to that of a monoclonal antibody (mab)-based ELISA using influenza ( flu ) A strains from 1968 to 1994. Specific PCR primers, generated to amplify the M2 gene region where amantadine resistance mutations occur, were tested over a wide range of strains. Direct sequencing of the PCR fragments was performed to confirm the presence of resistance mutations. RESULTS: The polyclonal antiserum-based ELISA detected antigens from all recent H1N1 strains and H3N2 strains tested at an inoculum dilution ten-fold lower than the mab-based ELISA. Primers for the detection of amantadine resistance mutations consistently amplified a wide range of strains. The direct sequencing of the RT-PCR amplicons generated, detected resistance mutations in reassortant and control viruses, the only strains found resistant by ELISA. All influenza ( flu ) A isolates (H3N2, H1N1) tested, except resistant controls and two reassortant viruses, were amantadine-sensitive as indicated by greater than 50% virus growth inhibition. CONCLUSIONS: Influenza ( Flu ) A virus susceptibility to amantadine could be detected by using an antiserum-based ELISA, offering a simple and more sensitive alternative to the mab-based assay. Coupled with direct sequencing of the M2 gene, it provides a reliable way to detect and confirm resistance in influenza ( flu ) isolates. However, no resistant clinical isolates were detected in the sample.

Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza ( flu ) virus or adenovirus.

During influenza ( flu ) virus infection, protein synthesis is maintained at high levels and a dramatic switch from cellular to viral protein synthesis occurs despite the presence of high levels of functional cellular mRNAs in the cytoplasm of infected cells (M. G. Katze and R. M. Krug, Mol. Cell. Biol. 4:2198-2206, 1984). To determine the step at which the block in cellular mRNA translation occurs, we compared the polysome association of several representative cellular mRNAs (actin, glyceraldehyde-3-phosphate dehydrogenase, and pHe7 mRNAs) in infected and uninfected HeLa cells. We showed that most of these cellular mRNAs remained polysome associated after influenza ( flu ) viral infection, indicating that the elongation of the proteins encoded by these cellular mRNAs was severely inhibited. Because the polysomes containing these cellular mRNAs did not increase in size but either remained the same size or decreased in size, the initiation step in cellular protein synthesis must also have been defective. Several control experiments established that the cellular mRNAs sedimenting in the polysome region of sucrose gradients were in fact associated with polyribosomes. Most definitively, puromycin treatment of infected cells caused the dissociation of polysomes and the release of cellular, as well as viral, mRNAs from the polysomes, indicating that the cellular mRNAs were associated with polysomes that were capable of forming at least a single peptide bond. A similar analysis was performed with HeLa cells infected by adenovirus, which also dramatically shuts down cellular protein synthesis. Again, it was found that most of the cellular mRNAs, which were translatable in reticulocyte extracts, remained associated with polysomes and that there was a combined initiation-elongation block to cellular protein synthesis. In cells infected by both adenovirus and influenza ( flu ) virus, influenza ( flu ) viral mRNAs were on larger polysomes than were several late adenoviral mRNAs with comparably sized coding regions. In addition, after influenza ( flu ) virus superinfection of cells infected by the adenovirus mutant dl331, a situation in which there is a limitation in the amount of functional initiation factor eIF-2 (M. G. Katze, B. M. Detjen, B. Safer, and R. M. Krug, Mol. Cell. Biol. 6:1741-1750, 1986), influenza ( flu ) viral mRNAs, but not late adenoviral mRNAs, were on polysomes. These results indicate that influenza ( flu ) viral mRNAs are better initiators of translation than are late adenoviral mRNAs.

Induction of antibody responses to influenza ( flu ) virus in human lymphocyte cultures. I. Role of interleukin 2.

The in vitro T cell-dependent antibody response of human lymphocytes to influenza ( flu ) virus X31 was used to study the role of T cell-derived lymphokines in antigen-specific responses. Supernatant from cultures of phytohaemagglutinin-stimulated, pooled human tonsil cells (PHA-MLR) was capable of replacing T cells and inducing T-depleted tonsil cells to secrete influenza ( flu )-specific antibody. The T cell-replacing activity of PHA-MLR supernatant co-purified with interleukin 2 (IL 2) on Ultrogel AcA54 gel filtration and reversed phase-high performance liquid chromatography. PHA-MLR supernatant and IL 2 also enhanced B cell proliferation induced by anti-mu or Staphylococcal aureus strain Cowan I (SAC). A murine monoclonal antibody directed against the human IL 2 receptor (Mab 2A3) was used to completely block the enhancement of influenza ( flu )-specific antibody production mediated by PHA-MLR supernatant, purified IL 2, and recombinant human IL 2. Mab 2A3 did not affect the T-independent B cell proliferation induced by anti-mu or SAC, but abrogated the enhancing effect of the PHA-MLR supernatant and IL 2 in this culture system. Immunofluorescence studies failed to demonstrate binding of Mab 2A3 to B cells activated by the X31 influenza ( flu ) virus and IL 2, or by SAC. By using Mab 2A3 to mask out IL 2 effects in the influenza ( flu )-specific culture system, no other B cell differentiating activities were revealed in supernatants from lymphocytic cultures stimulated with a variety of mitogens. Thus, our results indicate that the production of influenza ( flu )-specific antibodies by T-depleted human lymphocyte cultures is absolutely dependent on the presence of both antigen and IL 2.

The B allele of the NS gene of avian influenza ( Flu ) viruses, but not the A allele, attenuates a human influenza ( flu ) A virus for squirrel monkeys.

The nonstructural (NS) genes of avian influenza ( Flu ) A viruses have been divided into two groups on the basis of nucleotide sequence homology, which we have referred to here as alleles A and B. We sequenced the NS genes of eight additional avian influenza ( Flu ) A viruses in order to define the differences between these two alleles more thoroughly. Four of the viruses had NS gene sequences which resembled that of A/FPV/Rostock/34 and belonged to allele A while the other four viruses had NS gene sequences more similar to that of A/Duck/Alberta/76 and belonged to allele B. There was approximately 90% sequence homology within alleles and 72% homology between alleles. As previously reported the NS genes of human influenza ( flu ) A viruses belong to allele A. We constructed single gene avian-human reassortant influenza ( flu ) A viruses containing an allele A or B NS gene segment from an avian influenza ( Flu ) A virus and all other genes from a human influenza ( flu ) A virus and tested these reassortants for their ability to grow in the respiratory tract of a nonhuman primate. Reassortants containing an avian NS gene segment of allele B were significantly restricted in growth in the respiratory tract of squirrel monkeys while reassortants with an allele A NS gene segment were not. The divergent evolution of the B NS allele in birds may have resulted in gene products which do not function optimally in cooperation with genes from a human virus in viral replication in primate respiratory epithelium.

Influenza ( Flu ) B virus PB1 protein; nucleotide sequence of the genome RNA segment predicts a high degree of structural homology with the corresponding influenza ( flu ) A virus polymerase protein.

The complete nucleotide sequence of a cloned cDNA copy of the genome RNA segment encoding the influenza ( flu ) B/Lee/40 virus PB1 polymerase protein has been determined. The genome RNA segment is 2368 nucleotides in length and is capable of encoding a polymerase (PB1) protein of 752 amino acids with a calculated mol mass of 84,407 Da. As expected, the protein is highly basic with a net charge of +20 at pH 7.0. Sequence comparison between the influenza ( flu ) A and B virus PB1 proteins reveals that they share 61% amino acid homology. An internal hydrophobic domain and 90% of the proline residues found within the influenza ( flu ) A virus PB1 protein are conserved in the influenza ( flu ) B virus molecule. The influenza ( flu ) A and B virus PB1 proteins share the highest homology yet seen between proteins encoded by these disparate viruses. This remarkable conservation of primary structure argues for severe functional constraint on the evolution of this influenza ( flu ) virus polymerase protein.

Clinical evaluation of rapid diagnostic kit detecting separately influenza ( flu ) A and B viruses

The Directigen Flu A + B kit, a rapid diagnostic device for influenza ( flu ) virus A and B was evaluated. The nasopharyngeal aspirates were obtained from 239 patients who visited our hospital, between January and March, 2000, presenting flu-like symptoms. Influenza ( Flu ) virus AH1: 77 and AH3: 51 were isolated from 128 specimens and none from 111 specimens. Directigen Flu A + B showed 115 specimens positive and 106 specimens negative. The sensitivity and specificity of this kit were 89.8% (115/128) and 95.5% (106/111) compared with viral isolation. Agreement on positive and negative interpretations between Direction Flu A and this kit was 97.9% (234/239). In the evaluation of this kit for influenza ( flu ) B virus, 60 frozen nasopharyngeal aspirates collected from February to April, 1999 were used. The sensitivity and specificity of this kit were 88.9% (16/18) and 88.1% (37/42) compared with viral isolation. Agreement on positive and negative interpretations between FLU OIA and this kit was 91.7% (55/60). The Directigen A + B demonstrated sensitivity and specificity equivalent to the conventional kits in nasopharingeal aspirates. This kit can also differentiate influenza ( flu ) A and B viruses, a feature which is useful for treatment using anti-viral agents such as amantadine and neuraminidase inhibitor. To date, the kit is the most effective tool for the rapid diagnosis of influenza ( flu ).

Which response against the next influenza ( flu ) pandemic

An influenza ( flu ) pandemic is caused by the emergence or the reoccurrence as well as the explosive spreading of a new influenza ( flu ) A virus in an immunologically unprotected population. During this century, three severe pandemics occurred: the Spanish Flu in 1918, the Asian Flu in 1957 and the Hong Kong Flu in 1968. The first one was responsible for 20 millions deaths. A future pandemic is very likely to occur but we cannot predict when it will happen. The mechanism of emergence of a new virus is totally different of the one observed during the annual interpandemic outbreaks. Since the severity of a pandemic virus is unpredictable, action plans must be designed to avoid over-reaction to harmless influenza ( flu ) viruses and at the same time to be adequate in dealing with a threatened new pandemic. Thus these two situations need different control and prevention plans.

Molecular evolution of H6 influenza ( flu ) viruses from poultry in Southeastern China: prevalence of H6N1 influenza ( flu ) viruses possessing seven A/Hong Kong/156/97 (H5N1)-like genes in poultry.

The A/teal/Hong Kong/W312/97 (H6N1) influenza ( flu ) virus and the human H5N1 and H9N2 influenza ( flu ) viruses possess similar genes encoding internal proteins, suggesting that H6N1 viruses could become novel human pathogens. The molecular epidemiology and evolution of H6 influenza ( flu ) viruses were characterized by antigenic and genetic analyses of 29 H6 influenza ( flu ) viruses isolated from 1975 to 1981 and 1997 to 2000. Two distinct groups were identified on the basis of their antigenic characteristics. Phylogenetic analysis revealed that all H6N1 viruses isolated from terrestrial poultry in 1999 and 2000 are closely related to A/teal/Hong Kong/W312/97 (H6N1), and the nucleotide sequences of these viruses and of A/Hong Kong/156/97 (H5N1) were more than 96% homologous. The hemagglutinin (HA) of the 1999 and 2000 terrestrial viruses does not have multiple basic amino acids at the site of cleavage of HA1 to HA2; however, a unique insertion of aspartic acid in HA1 between positions 144 and 145 (H3 numbering) was found. The neuraminidase of these terrestrial H6N1 viruses has a deletion of 19 amino acids characteristic of A/Hong Kong/156/97 (H5N1). Evolutionary analysis suggested that these H6N1 viruses coevolved with A/quail/Hong Kong/G1/97-like H9N2 viruses and became more adapted to terrestrial poultry. These terrestrial 1999 and 2000 A/teal/Hong Kong/W312/97 (H6N1)-like viruses, along with the H9N2 viruses, could have been involved in the genesis of the pathogenic H5N1 influenza ( flu ) viruses of 1997. The presence of H6N1 viruses in poultry markets in Hong Kong that possess seven of the eight genes of the A/Hong Kong/156/97 (H5N1) virus raises the following fundamental questions relevant to influenza ( flu ) pandemic preparedness: could the pathogenic H5N1 virus reemerge and could the H6N1 viruses directly cross the species barrier to mammals?