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Type A influenza isolates possessing the hemagglutinin (HA)neuraminidase (NA) combination of H1N1 have been of great interest to both public health and veterinary scientists and officials since the occurrence of the “Spanish Flu” pandemic and the emergence of the classical H1N1 “swine influenza” in 1918. H1N1 type A influenza viruses have been recovered from wild and domestic birds, swine and humans but these isolates show relatively strong predilections for their respective hosts. However, this host specificity is not absolute and H1N1 type A influenza viruses have been show to cross interspecies barriers between turkeys and swine, humans and swine, avian species and swine, and wild and domestic birds. (Wuethrich) (Dowdle and M.A.Hattwick) (Lipkind) (Johnson) The pathogenicity and virulence properties of type A influenza viruses are dependent on the interactions and relationships among the eight negative-sense RNA gene segments making up the genomic constellation and the eleven proteins coded for by these genomic segments. These genomic segments theoretically function independently and recent studies indicate that in nature the genomic constellations of waterfowl-origin type A influenza viruses are formed with no clear pattern of gene segment association. This raises the possibility that genomic segments of H1N1 influenza A viruses could be moving between wild birds, poultry, swine and mammalian hosts via genetic reassortment and this movement could go undetected if the isolates are only examined using standard serological tests, i.e., hemagglutination- and neuraminidase-inhibition tests which are based on the antigenic properties of the two surface glycoproteins. Waterfowl-origin H1N1 type A influenza viruses in our repository appeared to demonstrate differences in infectivity and hemagglutinin titers when propagated in embryonating chicken eggs. There are several possible explanations for these apparent differences. The possible explanations include, but are not limited to 1) inhibitors and incomplete virus particles in original samples and first egg passage fluid which would inhibit complete, viable virus particles from attaching to host receptor sites. 2) Varying viral concentrations in original samples could affect the final HA titer and EID50 because inoculums with higher concentrations of virus would have more particles replicating and available to attach to red blood cells in a hemagglutination test. 3) Point mutations in the genomic segments which lead to amino acid substitutions that could change the phenotypic properties of an isolate. 4) Lastly, differences in genomic constellations (all eight RNA segments in the genome) resulting from genomic reassortments with genes from other lineages of water-fowl origin type A influenza viruses and even viruses maintained in lower mammals and humans which could be influencing the phenotypic differences. The hypothesis for this investigation is that, under the conditions of this study, the growth rates, hemagglutination titers, and maximum infectiousness of four low pathogenic waterfowl-origin (WFO) H1N1 type A influenza virus (AIV) isolates selected for this study are influenced by variations in nucleotide/amino acid sequences due to prior genetic reassortment and/or point mutations. The influences of inhibitors and incomplete virus particles were minimized by limiting dilution passages in embryonating chicken eggs. Varying virus concentrations in each inoculum were accounted for by standardizing inoculums at approximately 1x102 EID50/1ml. Upon the completion of genomic analysis it was determined that all eight genomic segments in each isolate were waterfowl-origin, even though there was up to a 34.5% difference in amino acid sequence homology between the isolates. This eliminated the possibility that genetic reassortment with domestic poultry- and mammalian-origin influenza viruses was responsible for the phenotypic variations. Therefore, it was concluded that the phenotypic differences observed in this study were due to the nucleotide differences resulting from point mutations and prior accumulating amino acid substitutions observed among the isolates, and not due to genomic reassortment with H1N1 viruses from domestic poultry, swine, or humans. Interestingly, the amino acid differences were not associated with previously reported amino acid substitutions affecting replication of human origin influenza viruses in embryonating chicken eggs. |
