Genetic and molecular characterisation of non-tissue culture adapted influenza B viruses detected in Singapore between 2004-2009
Muhammad Raihan Jumat
Date of Issue2014
School of Biological Sciences
In Singapore, both influenza A and B viruses circulate in significant proportions throughout the year, with bi-annual peaks corresponding to the monsoon seasons. This statistic is not shared in most temperate countries where influenza is only prevalent during winter. Exploiting this unique situation in Singapore, we studied the characteristics of non-tissue culture adapted influenza B clinical specimens collected from SAF servicemen between the years of 2004 and 2009. A total of 81 clinical specimens were received and 66 tested positive for influenza B upon diagnostic PCR. Growth kinetics of these clinical specimens could not be studied as none of these specimens could replicate in tissue culture. Forty-six of these clinical specimens yielded sequence information for further analysis. Lineage identity of these specimens were determined through phylogenetic analysis, followed by a comparison of the amino acid sequences between the HA, NA, NB, NS1, NEP and BM2 proteins. Molecular cloning of the NB, NS1 and BM2 genes of representative strains were carried out to determine if differences in sequence would translate into phenotypic differences. Phylogenetic analysis show that majority of the specimens were re-assortants with the HA belonging to the Victoria lineage and the NA from the Yamagata lineage. Only one specimen had both these gene segments from the Yamagata lineage. This finding has resulted in us determining several vaccine mismatches over the six years in focus. The NS1 phylogenetic tree showed that seven of the specimens isolated in 2004 clustered closely to B/Lee/40 than the rest. This finding provides an interesting look into the separation of the influenza B lineages. Analysis of the amino acid sequences reveal that majority of the clinical specimens harboured a glycosylation site at position 211 of the HA protein, which was previously not dominant. No mutations conferring drug resistance were noticed among the HA and NA proteins analysed. The 7 specimens clustering with B/Lee/40 in the NS phylogenetic tree had almost identical amino acid sequences to B/Lee/40 NS1 and NEP proteins, differing from the other clinical specimens. We found an inability of these clinical specimens to replicate well in tissue culture, a trait shared with the reference strain B/Lee/40. In an attempt to exploit the differences in amino acid sequences within the clinical specimens, the NB, BM2 and NS1 genes of representative sequences were cloned into a mammalian expression xvii vector. Basic molecular characterisation indicates that the NB protein, which has an unknown role, localised primarily in the cis-Golgi and is present throughout the Golgi. This is similar to the staining pattern displayed by the BM2 protein, suggesting a similarity in the function. SDS-PAGE analysis reveal that both these proteins are modified by the cell and further elucidation is required to determine the kinetics and the function of these modifications. The bulk of the work presented in this thesis revolves around the molecular characterisation of the influenza B NS1 protein. Two main ‘phenotypes’ were observed in respect to cellular localisation; speckled and un-speckled. Two of the strains did not display localisation with nuclear speckles while three did. The introduction of mutations as well as expression of the different domains of the protein did not clarify which residues were involved in the dynamics of this nuclear speckle interaction. Four of the specimens analysed presented a second, smaller peptide on SDS-PAGE analysis. Site-directed mutagenesis revealed that an aspartic acid residue at position 92, instead of an asparagine seems to be responsible for the presence of this smaller peptide. Even so, the mechanism yielding this peptide is still unknown since caspase and MMP inhibitors did not have an effect in its expression. Further elucidation has to be carried out to determine both the mechanism of expression as well as its function in viral replication. The inability of the clinical specimens collected in this study prompts a detailed exploration into the choice of cell-culture systems in passaging influenza B clinical specimens. This may also reflect the lack of understanding of influenza biology, which has been focused primarily on lab-adapted strains instead of naturally circulating strains. The sequence information of the analysed specimens reveal that majority of the clinical specimens sequenced in this study were re-assortants with their HA from the Victoria lineage and the NA from the Yamagata lineage. Only 1 specimen proved to have both these glycoproteins from the Yamagata lineage. Sequence analysis of the HA protein show that a glycosylation site at position 211 is now prevalent amongst circulating strains and no neuraminidase inhibitor mutations were observed in the HA and NA genes. Cloning of the NS1 gene showed that some of the specimens had a nuclear speckled localisation while other did not. The exact domain within the protein responsible for this was not determined in this study and further probing has to be xviii carried out. A smaller peptide, p23, was expressed in 4 of the 6 cloned NS1 genes. This is the first time the expression of p23 has been documented. The amino acids at the interface of the RNA-binding domain and the linker were crucial for its expression or silencing. The exact mechanism of its expression is yet to be elucidated.
DRNTU::Science::Biological sciences::Molecular biology