Alexander I. Klimov

This chapter describes some commonly used methods of influenza virus titration, antigenic characterization, and serological methods by antibody detection. These methods are essential not only for virus characterization but also for identifying new antigenic variants, vaccine strain selection, and sero-epidemiologic studies of influenza virus transmission and prevalence. Virus titration methods such as the hemagglutination assay, 50% egg or tissue culture infectious dose, and plaque assay are employed to determine the amount of virus particles in a sample. The hemagglutination inhibition assay is a reliable, relatively simple and inexpensive technique to antigenically characterize isolates of influenza viruses. Serological methods such as virus neutralization and hemagglutination inhibition are the fundamental tools used in sero-epidemiologic studies of influenza virus transmission and prevalence and in the evaluation of vaccine immunogenicity. While serological methods rarely yield an early diagnosis of acute influenza virus infection, well-timed, paired acute, and convalescent serum samples may establish the diagnosis of a recent influenza infection even when attempts to detect the virus are negative.

For the development of live attenuated influenza vaccine (LAIV) against influenza virus strains with pandemic potential, method of classic genetic reassortment of donor of attenuation A/Leningrad/134/17/57 (H2N2) [Len/17] with avian apathogenic influenza viruses of different subtypes was used. Strain with genome formula 6:2, which contains HA and NA genes from avian apathogenic virus A/wild duck/Netherlands/12/00 (H7N3) [N7N3-wt] and 6 other genes--from Len/17, was studied. Reassortant strain A/17/ wild duck/Netherlands/00/84 (H7N3) [Lenl7/ H7] exhibited ts- and ca- phenotype specific for cold-adapted strains. Reassortant was identical on antigenic profile to parent avian virus H7N3-wt. Like cold-adapted donor strain Len/17, Len17/H7 was attenuated for chickens, whereas wild-type parent strain was lethal in 60% of birds after its intravenous challenge. Reassortant strain Len17/H7 was attenuated during intranasal inoculation of 6 EID50 to white mice, which was confirmed by absence of its isolation from the lungs, actively reproduced on nasal mucosa and stimulated specific systemic and local antibody response.

Background: Influenza A viruses of domestic birds originate from the natural reservoir in aquatic birds as a result of interspecies transmission and adaptation to new host species. We previously noticed that influenza viruses isolated from distinct orders of aquatic and terrestrial birds may differ in their fine receptor-binding specificity by recognizing the structure of the inner parts of Neu5Acα2-3Gal-terminated sialyloligosaccharide receptors. To further characterize these differences, we studied receptor-binding properties of a large panel of influenza A viruses from wild aquatic birds, poultry, pigs and horses. Results: Using a competitive solid-phase binding assay, we determined viral binding to polymeric conjugates of sialyloligosaccharides differing by the type of Neu5Acα-Gal linkage and by the structure of the more distant parts of the oligosaccharide chain. Influenza viruses isolated from terrestrial poultry differed from duck viruses by an enhanced binding to sulfated and/or fucosylated Neu5Acα2-3Gal-containing sialyloligosaccharides. Most of the poultry viruses tested shared a high binding affinity for the 6-sulfo sialyl Lewis X (Su-SLe^x). Efficient binding of poultry viruses to Su-SLe^x was often accompanied by their ability to bind to Neu5Acα2-6Gal-terminated (human-type) receptors. Such a dual receptor-binding specificity was demonstrated for the North American and Eurasian H7 viruses, H9N2 Eurasian poultry viruses, and H1, H3 and H9 avian-like virus isolates from pigs. Conclusion: Influenza viruses of terrestrial poultry differ from ancestral duck viruses by enhanced binding to sulfated and/or fucosylated Neu5Acα2-3Gal-terminated receptors and, occasionally, by the ability to bind to Neu5Acα2-6Gal-terminated (human-type) receptors. These findings suggest that the adaptation to receptors in poultry can enhance the potential of an avian virus for avian-to-human transmission and pandemic spread.

Classical genetic reassortant techniques were used to have a cold-adapted (ca) reassortant A/17/Duck/Potsdam/86/92 (H5N2) that inherited the hemagglutinin (HA) gene from the nonpathogenic avian virus A/Duck/Potsdam/ 1402-6186 (H5N2) and the genes of neuraminidase (NA) and non-glycated proteins from the ca attenuation donor A/Leningrad/134/17/57 (H2N2). All experiments were performed under increased biological protection (BSV-3+). The reassortant and parent H5N2 virus were non-pathogenic to Balb/c mice, the reassortant replication in the murine nasal passages (3.5 Ig EID50/ml) being higher than that in the lung (2.1 lg EID50/ml). Intranasal inoculation of mice with reassortant A/17/Duck/Potsdam/86/92 caused an immune response to both homological H5N2 virus and antigenically differing variants of influenza A (H5N1) virus isolated from humans in 1997 and 2003. The mice intranasally immunized with the ca reassortant were protected against fatal infection with the highly pathogenic A/Hong Kong/483/9797 (H5N1) virus and against infection with A/Hong Kong/213/03(H5N1) virus (80 and 100%, respectively).

A simple and sensitive method has been developed to determine the genome composition of the reassortant on the basis of B/USSR/60/69 by the restrictase analysis of DNA copies of RNA sites containing the nucleotide replacements typical of B/USSR/60/69.

The authors examined a role of some mutated A/Leningrad/134/17/57(H2N2) virus genes in the realization of growth characteristics. The latter of single gene reassortants (SGRs) (PB2, PB1, PA, M, and NS), epidemic virus and attenuation donor were assessed by infecting MDCK cells and hen embryos at a low inoculation index. Viral replication in the hen embryos and cultured tissue was compared at 34 degrees C. The viruses and reassortants tested showed a high growth capacity in the hen embryos (9.5-10.5 Ig TCID50). The growth curves of viruses were studied on the cultured MDCK cells at a low inoculation index indicated that Len/17 and the single gene reassortants M and NS had the highest growth capacity. At the same time the growth of both PB1 and PB2 SGRs was less extensive. The reproduction of PB2 SGR was 100-1000 times less than that of other viruses tested. M, NS, and PA gene mutations did not affect viral growth in hen embryos and cultured tissue while PB2 gene mutation and its constellations with other genes caused a reduction in viral growth in the cultured tissue.

Production lots of a live influenza vaccine made of strains A/47/T (N1H1), A/47/6/2 (H3N2), and B/60/32 were used for vaccination of 3663 children aged from 5 to 14 years inoculated twice with monovaccines, a trivaccine made of the above strains, or placebo. Both mono- and polyvaccine were practically areactogenic. An average per cent of subjects with a significant rise in antibody titres to the respective three antigens was 60%. The efficacy of the vaccination was 31.0-42.8% for monopreparations and 36.3% for the trivaccine. The studies showed the possibility and expedience of using for children the live influenza vaccine in the form of a polyvalent preparation including current influenza type A and B viruses.