PA-X is an avian virulence factor in H9N2 avian influenza virus

Clements, Anabel L.; Peacock, Thomas P.; Sealy, Joshua E; Hussain, Saira; Sadeyen, Jean-Rémy; Shelton, Holly; Digard, Paul; Iqbal, Munir

doi:10.1101/2020.05.25.114876

Cited by 4 publications

(14 citation statements)

References 37 publications

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“…All data were normalized to a control where segment 3 was substituted for an empty vector. UDL-01 WT segment 3 significantly reduced levels of β-gal compared to the empty vector control indicating robust shutoff activity ( Figure 5A), as shown previously (34). In contrast WF10 WT segment 3 displayed no detectable shutoff, giving equivalent β-gal signal to the empty on November 1, 2020 by guest http://jvi.asm.org/ Downloaded from vector control.…”

Section: Pa-xsupporting

confidence: 81%

“…We initially investigated the combined effect of position and FS mutations on host shutoff in mammalian and avian cells ( Figure 6A,B). For UDL-01 WT introduction of either the E26K or FS mutation individually or together ablated shutoff activity, indicating as we and others have shown, that segment 3 shutoff activity maps to PA-X (19,34,35,37,38).…”

Section: Downloaded Fromsupporting

confidence: 76%

“…The influenza virus accessory protein, PA-X, has been described in multiple studies as a virulence factor in avian influenza viruses (including H9N2 viruses) that can affect disease outcome in mammals or birds (22,24,26,34). Although other studies have found PA-X expression can lead to an attenuated phenotype, particularly in highly pathogenic H5N1 viruses (23,25).…”

Section: Discussionmentioning

confidence: 99%

“…reporter assay was used to test the ability of the PA-X proteins from these viruses to cause host cell shutoff (19,34). Briefly, cells were co-transfected with expression plasmids containing the different segment 3s along with a β-gal reporter plasmid, followed by enzymatic readout of β-gal activity to give a measure of host gene expression in the transfected cells and thus the ability of the different segment 3 plasmids to cause host shutoff.…”

Section: Pa-xmentioning

confidence: 99%

“…The changes at PA position 26 are responsible for attenuation of WF10 in vitro and in vivo; these correlated better with the in vitro host cell shutoff activity of PA-X than with polymerase activity. To investigate whether the E26K polymorphism exerted its in vivo phenotypic effect via PA-X rather than through PA, we introduced a well characterized set of nucleotide substitutions into the frameshift site (FS mutant) of PA which has previously been shown to inhibit expression of PA-X (19,34,35). We further combined this FS mutation with the reciprocal mutants at position 26.…”

Section: Differences In Pa-x Alone Are Not Responsible For the Attenumentioning

confidence: 99%

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Contribution of Segment 3 to the Acquisition of Virulence in Contemporary H9N2 Avian Influenza Viruses

Clements

Sealy

Peacock

et al. 2020

J Virol

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H9N2 avian influenza viruses circulate in poultry throughout much of Asia, the Middle East and Africa. These viruses cause huge economic damage to poultry production systems and pose a zoonotic threat both in their own right as well as in the generation of novel zoonotic viruses, for example, H7N9. In recent years it has been observed that H9N2 viruses have further adapted to gallinaceous poultry, becoming more highly transmissible and causing higher morbidity and mortality. Here, we investigate the molecular basis for this increased virulence, comparing a virus from the 1990s and a contemporary field strain. The modern virus replicated to higher titres in various systems and this difference mapped to a single amino acid polymorphism at position 26 of the endonuclease domain shared by the PA and PA-X proteins. This change was responsible for increased replication and higher morbidity and mortality rates along with extended tissue tropism seen in chickens. Although the PA K26E change correlated with increased host cell shutoff activity of the PA-X protein in vitro, it could not be overridden by frameshift site mutations that block PA-X expression and therefore increased PA-X activity could not explain the differences in replication phenotype. Instead, this indicates these differences are due to subtle effects on PA function. This work gives insight into the ongoing evolution and poultry adaptation of H9N2 and other avian influenza viruses and helps us understand the striking morbidity and mortality rates in the field, as well as rapidly expanding geographical range seen in these viruses. Importance Avian influenza viruses, such as H9N2, cause huge economic damage to poultry production worldwide and are additionally considered potential pandemic threats. Understanding how these viruses evolve in their natural hosts is key to effective control strategies. In the Middle East and South Asia an older H9N2 virus strain has been replaced by a new reassortant strain with greater fitness. Here we take representative viruses and investigate the genetic basis for this ‘fitness'. A single mutation in the virus was responsible for greater fitness, enabling high growth of the contemporary H9N2 virus in cells, as well as in chickens. The genetic mutation that modulates this change is within the viral PA protein, a part of the virus polymerase gene that contributes in viral replication as well as contribute in the virus accessory functions – however, we find that the fitness effect is specifically due to changes in the protein polymerase activity.

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Section: Pa-xsupporting

confidence: 81%

Section: Downloaded Fromsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Section: Pa-xmentioning

confidence: 99%

Section: Differences In Pa-x Alone Are Not Responsible For the Attenumentioning

confidence: 99%

See 3 more Smart Citations

Contribution of Segment 3 to the Acquisition of Virulence in Contemporary H9N2 Avian Influenza Viruses

Clements

Sealy

Peacock

et al. 2020

J Virol

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Contribution of segment 3 to the acquisition of virulence in contemporary H9N2 avian influenza viruses

Clements

Sealy

Peacock

et al. 2020

Preprint

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H9N2 avian influenza viruses circulate in poultry throughout much of Asia, the Middle East and Africa. These viruses cause huge economic damage to poultry production systems and pose a zoonotic threat both in their own right as well as in the generation of novel zoonotic viruses, for example H7N9. In recent years it has been observed that H9N2 viruses have further adapted to poultry, becoming more highly transmissible and causing higher morbidity and mortality. Here, we investigate the molecular basis for this increased virulence, comparing a virus from the 1990s and a contemporary field strain. The modern virus replicated to higher titres in various systems and this difference mapped to a single amino acid polymorphism at position 26 of the endonuclease domain shared by the PA and PA-X proteins. This change was responsible for the virulent phenotype and extended tissue tropism seen in chickens. Although the PA K26E change correlated with increased host cell shutoff activity of the PA-X protein in vitro, it could not be overridden by frameshift site mutations that block PA-X expression and therefore increased PA-X activity could not explain the differences in replication phenotype. Instead, this indicates these differences are due to subtle effects on PA function. This work gives insight into the ongoing evolution and poultry adaptation of H9N2 and other avian influenza viruses and helps us understand the soaring morbidity and mortality rates in the field, as well as rapidly expanding geographical range seen in these viruses.Author SummaryAvian influenza viruses, such as H9N2, cause huge economic damage to poultry production worldwide and are additionally considered potential pandemic threats. Understanding how these viruses evolve in their natural hosts is key to effective control strategies. In the Middle East and South Asia an older H9N2 virus strain has been replaced by a new reassortant strain with greater fitness. Here we take representative viruses and investigate the genetic basis for this ‘fitness’. A single mutation in the virus was responsible for greater fitness, enabling high growth of the contemporary H9N2 virus in cells, as well as in chickens. The genetic mutation that modulates this change is within the viral PA protein, a part of the virus polymerase gene that contributes in viral replication as well as contribute in the virus accessory functions – however we find that the fitness effect is specifically due to changes in the protein polymerase activity.

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The origin of internal genes contributes to the replication and transmission fitness of H7N9 avian influenza virus

James

Bhat

Walsh

et al. 2022

Preprint

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H9N2 avian influenza viruses (AIVs) have donated internal gene segments during the emergence of zoonotic AIVs, including H7N9. We used reverse genetics to generate three reassortant viruses (2:6 H7N9) which contained the Haemagglutinin and Neuraminidase from Anhui/13 (H7N9) and the six internal gene segments from H9N2 AIVs of G1-like or BJ94-like lineages enzootic in different geographic regions in Asia. Infection of chickens with the 2:6 H7N9 containing internal gene segments from G1-like H9N2 conferred attenuation in vivo, with lower shedding and reduced transmission to contact chickens. However, possession of BJ94-like H9N2 internal gene segments resulted in more rapid transmission and significantly elevated cloacal shedding compared to the parental Anhui/13 H7N9. In vitro analysis also showed that the 2:6 H7N9 having BJ94-like internal genes had significantly increased replication compared to the Anhui/13 H7N9 in chicken cells. In vivo co-infection experiments followed, where chickens were co-infected with pairs of Anhui/13 H7N9 and one of each of the three 2:6 H7N9 reassortants. During ensuing transmission events, the Anhui/13 H7N9 virus outcompeted 2:6 H7N9 with internal gene segments of BJ94-like or G1-like H9N2 viruses. Co-infection did lead to the emergence of novel reassortant genotypes that were transmitted to contact chickens. Some of the reassortant viruses had a greater replication in chicken and human cells compared to the progenitors. We demonstrated that the internal gene cassette determines the transmission fitness of H7N9 viruses in chickens and the reassortment events can generate novel H7N9 genotypes with increased virulence in chickens and enhanced zoonotic potential.

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PA-X is an avian virulence factor in H9N2 avian influenza virus

Cited by 4 publications

References 37 publications

Contribution of Segment 3 to the Acquisition of Virulence in Contemporary H9N2 Avian Influenza Viruses

Contribution of Segment 3 to the Acquisition of Virulence in Contemporary H9N2 Avian Influenza Viruses

Contribution of segment 3 to the acquisition of virulence in contemporary H9N2 avian influenza viruses

The origin of internal genes contributes to the replication and transmission fitness of H7N9 avian influenza virus

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