21 research outputs found
H5N1 Clade 2.2 Polymorphism Tracing Identifies Influenza Recombination and Potential Vaccine Targets
Highly pathogenic Influenza A H5N1 was first identified in Guangdong Province in 1996, followed by human cases in Hong Kong in 1997 1. The number of confirmed human cases now exceeds 300 and the associated Case Fatality Rate exceeds 60% 2. The genetic diversity of the serotype continues to increase. Four distinct clades or sub-clades have been linked to human cases 3.4. The gradual genetic changes identified in the sub-clades have been attributed to copy errors by viral encoded polymerases that lack an editing function, thereby resulting in antigenic drift 5. We traced polymorphism acquisition in Clade 2.2 sequences. We report here the concurrent acquisition of the same polymorphism by multiple, genetically distinct, Clade 2.2 sub-clades in Egypt, Russia and Ghana. These changes are not easily explained by the current theory of “random mutation” through copy error, and are more easily explained by recombination with a common source. This conclusion is supported by additional polymorphisms shared by Clade 2.2 isolates in Egypt, Nigeria and Germany including aggregation of regional polymorphisms from each of these areas into a single Nigerian human hemagglutinin gene
Concurrent Acquisition of a Single Nucleotide Polymorphism in Diverse Influenza H5N1 Clade 2.2 Sub-clades
Highly pathogenic Influenza A H5N1 was first identified in Guangdong Province in 1996, followed by human cases in Hong Kong in 1997. The number of confirmed human cases now exceeds 300, and the associated Case Fatality Rate exceeds 60%. The genetic diversity of the serotype continues to increase. Four distinct clades or sub-clades have been linked to human cases. The gradual genetic changes identified in the sub-clades have been attributed to copy errors by viral encoded polymerases that lack an editing function, thereby resulting in antigenic drift. We report here the concurrent acquisition of the same polymorphism by multiple, genetically distinct, clade 2.2 sub-clades in Egypt, Russia, and Ghana. These changes are not easily explained by the current theory of “random mutation” through copy error, and are more easily explained by recombination with a common source. This conclusion is supported by additional polymorphisms shared by clade 2.2 isolates in Egypt and Germany
Aggregation of Single Nucleotide Polymorphisms in a Human H5N1 Clade 2.2 Hemagglutinin
The evolution of H5N1 has attracted significant interest 1-4 due to linkages with avian 5,6 and human infections 7,8. The basic tenets of influenza genetics 9 attribute genetic drift to replication errors caused by a polymerase complex that lacks a proof reading function. However, recent analysis 10 of swine influenza genes identifies regions copied with absolute fidelity for more than 25 years. In addition, polymorphism tracing of clade 2.2 H5N1 single nucleotide polymorphisms identify concurrent acquisition 11 of the same polymorphism onto multiple genetic backgrounds in widely dispersed geographical locations. Here we show the aggregation of regional clade 2.2 polymorphisms from Germany, Egypt, and sub-Sahara Africa onto a human Nigerian H5N1 hemagglutinin (HA), implicating recombination in the dispersal and aggregation of single nucleotide polymorphisms from closely related genomes
Aggregation of Single Nucleotide Polymorphisms in a Human H5N1 Clade 2.2 Hemagglutinin
The rapid evolution of the H5N1 serotype of avian influenza has been explained by a mechanism involving the selection of single nucleotide polymorphisms generated by copy errors. The recent emergence of H5N1 Clade 2.2 in fifty countries, offered a unique opportunity to view the acquisition of new polymorphism in these evolving genomes. We analyzed the H5N1 hemagglutinin gene from a fatal human case from Nigeria in 2007. The newly emerged polymorphisms were present in diverse H5N1 isolates from the previous year. The aggregation of these polymorphisms from clade 2.2 sub-clades was not supported by recent random mutations, and was most easily explained by recombination between closely related sequences
Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry
Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology.Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999-2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005-2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution.Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance
Pathogenic mechanisms of Campylobacter jejuni Characterization and identification of the role of CADF in Campylobacter mediated enteritis
Thesis (Ph.D.), Microbiology, Washington State Universit
Fibronectin-Facilitated Invasion of T84 Eukaryotic Cells by Campylobacter jejuni Occurs Preferentially at the Basolateral Cell Surface
Previous studies have indicated that the ability to bind to fibronectin is a key feature in successful cell invasion by
Campylobacter jejuni
. Given the spatial distribution of fibronectin and the architecture of the epithelium, this suggests the possibility that
C. jejuni
cell invasion might preferentially occur at the basolateral cell surface. To test this hypothesis, we examined the interaction of
C. jejuni
with T84 human colonic cells. When grown under the appropriate conditions, T84 cells form a polarized cell monolayer.
C. jejuni
translocation of a T84 cell monolayer appeared to occur via a paracellular (extracellular) route as opposed to a transcellular (intracellular) route based on the finding that a
C. jejuni
noninvasive mutant translocated as efficiently as its isogenic parent. Additional studies revealed that two distinct
C. jejuni
wild-type isolates could compete with one another for host cell receptors, whereas a
C. jejuni
fibronectin-binding-deficient mutant could not compete with a wild-type isolate for host cell receptors. Further,
C. jejuni
adherence and internalization were significantly inhibited by antifibronectin antibodies but only when cells were first treated with EGTA to expose basolateral cell surfaces. Together, these results support the theory that
C. jejuni
invasion occurs preferentially at the basolateral surface of eukaryotic cells
Secretion of Virulence Proteins from Campylobacter jejuni Is Dependent on a Functional Flagellar Export Apparatus
Campylobacter jejuni
, a gram-negative motile bacterium, secretes a set of proteins termed the
Campylobacter
invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (
flgB
and
flgC
), hook (
flgE2
), and filament (
flaA
and
flaB
) genes, as well as in genes whose products are essential for flagellar protein export (
flhB
and
fliI
). While mutations that affected filament assembly were found to be nonmotile (Mot
−
) and did not secrete Cia proteins (S
−
), a
flaA
(
flaB
+
) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot
−
, S
+
). Complementation of a
flaA flaB
double mutant with a shuttle plasmid harboring either the
flaA
or
flaB
gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the
C. jejuni
mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the
C. jejuni
Cia proteins are secreted from the flagellar export apparatus