Die Rolle des aviären PB1 Gens bei der Entstehung pandemischer Influenza-A-Viren
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Philipps-Universität Marburg
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Abstract
The 1957 and 1968 pandemic influenza viruses emerged via reassortment and contained HA
and PB1 gene segments of avian origin. It is well known that the acquisition of an antigenically novel avian HA allowed the emerging virus to overcome pre-existing immunity in the human population. At the same time, the biological significance of the presence of the avian PB1 in pandemic influenza viruses remains elusive. In this work we wished to address this longstanding question, and to determine whether avian PB1 provided pandemic influenza viruses with some replicative advantage in humans.
In the first part of the thesis, I modelled the reassortment event that led to the emergence of the 1968 pandemic influenza virus from its avian and human precursors. Using human H2N2 virus A/California/1/66 (Cal66), two recombinant Cal66 virus pairs were generated, in which the human-origin PB1 and/or HA gene segments were substituted by corresponding avian-like gene segments of the pandemic strain A/Hong Kong/1/68 (HK68). To test, whether avian PB1 facilitated viral replication and transmission of the pandemic virus, competitive replication experiments in human bronchial epithelial cells (Calu-3), as well as competitive contact transmission experiments in guinea pigs were performed. Viral competition in Calu-3 cells was studied by making consecutive passages starting from a 1:1 mixture of viruses and
determining composition of the passage 3 material by sequencing. In addition competitive
rescue experiments were performed by transfecting cells with a whole set of reverse genetics plasmids which included a 1:1 mixture of PB1Cal and PB1HK plasmids. The composition of rescued virus mixtures was determined by sequencing. To study contact transmission efficiency of the viruses, a group of four guinea pigs was inoculated with an equal mixture of viruses. One day post-infection (p.i.) four naïve animals were co-housed with infected animals and at different days p.i. nasal washes were taken and characterized by sequencing.
These studies demonstrated, that avian PB1 enhances viral replication independently of the
origin of the HA gene segment, possibly due to an enhanced viral polymerase activity.
The human-adapted avian PB1 of the 1968 pandemic virus differed from the avian consensus sequence by three amino-acid substitutions. In the second part of the thesis, I determined whether these three substitutions affected viral polymerase fidelity during the emergence of the pandemic virus. To this end, I generated recombinant HK68 viruses, which contained either individual mutations back to the avian consensus sequence or a combination of all three mutations.
The mutation frequency of the triple mutant was slightly enhanced in comparison to that of
the original HK68 virus. By contrast, two of the three tested single mutants (rHK-PB1HK-V212L
and rHK-PB1HK-K327R) showed a significantly enhanced mutation frequency. These results
suggest that mutation(s) in PB1 that emerged during the avian-to-human transmission could
decrease fidelity of the polymerase, and that the polymerase fidelity was later restored by
additional mutation(s). This scenario would agree with the generally accepted hypothesis that a temporary decrease in fidelity could provide selective advantage for zoonotic transmission and that the fidelity is restored as soon as the virus becomes adapted to a new host species.
In summary, data obtained in this thesis demonstrate for the first time that the avian-origin
PB1 of the pandemic 1968 virus served to promote viral replication and transmission. In
addition, our results suggest that a temporarily reduced polymerase fidelity could have
facilitated human adaptation of the emerging pandemic virus.
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Created: 2014Issued: 2015-05-28
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
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DoctoralThesis
Keywords
virale Transmissionviral transmissionreplication efficiencyPB1 geneReplikationseffizienzPB1 Gen
DFG-subjects
PandemieGrippe
DDC-Numbers
570
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Wendel, Isabel (1062555163): Die Rolle des aviären PB1 Gens bei der Entstehung pandemischer Influenza-A-Viren. : Philipps-Universität Marburg 2015-05-28. DOI: https://doi.org/10.17192/z2014.0465.
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This item has been published with the following license: In Copyright