H9 avian influenza reassortant with engineered polybasic cleavage site displays a highly pathogenic phenotype in chicken

Gohrbandt, Sandra; Veits, Jutta; Breithaupt, Angele; Hundt, Jana; Teifke, Jens Peter; Stech, Olga; Mettenleiter, Thomas C.; Stech, Jürgen

doi:10.1099/vir.0.031591-0

Cited by 49 publications

(64 citation statements)

References 56 publications

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“…The involvement of such cryptic virulence determinants has been supported by results from a number of studies (8,25,29).…”

Section: Discussionmentioning

confidence: 56%

“…To illustrate, a H5N2 virus isolated from a broiler flock and two live bird markets in Texas in 2004 was phenotypically low pathogenic, although possessing the HA 0 cleavage site PQRKKR/ GLF and hence genotypically HPAIV (7). Discordant results between molecular and in vivo tests used to characterize virus patho- genicity were retrospectively recognized for other H5 field virus isolates (8,9), as well as non-H5/H7 viruses that had been engineered to contain polybasic HA 0 cleavage sites (25,26,27). For example Stech et al (26) showed that inserting a polybasic cleavage site identical to that of HPAI A/chicken/Italy/8/98 (H5N2) into the HA of the LPAI A/duck/Ukraine/1/63 (H3N8) virus was not sufficient to immediately transform it into a highly pathogenic virus, despite the fact that it was able to replicate in tissue culture in the absence of exogenous trypsin.…”

Section: Discussionmentioning

confidence: 99%

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Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA ₀ Cleavage Site

Diederich

Berhane

Embury-Hyatt

et al. 2015

J Virol

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Although a polybasic HA 0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA 0 cleavage site have been identified that are low pathogenic in chickens. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA 0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity rH5N3 increased its virulence, transmission to naive contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype. IMPORTANCETo date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin-neuraminidase combinations, may have unpredictable effects on virulence and transmission characteristics of a virus. Our data show the importance of the neuraminidase in complementing a polybasic HA 0 cleavage site. Furthermore, it demonstrates that adaptive changes selected for during the course of virus evolution can result in unexpected traits such as antiviral drug resistance.A vian influenza viruses (AIV) belong to the genus Influenzavirus A in the family Orthomyxoviridae. Although wild waterfowl are considered the natural reservoir for all influenza A viruses, sporadic transmission to poultry and mammals can result in viruses that are adapted to a new host. The roles that the different viral genes play in this process, especially as it relates to tropism and virulence for domestic poultry species, are still not well understood.Influenza A viruses contain a negative-sense, single-stranded, eight-segmented RNA genome that codes for at least 11 proteins. Viruses are categorized based on the hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins. Sixt...

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“…The involvement of such cryptic virulence determinants has been supported by results from a number of studies (8,25,29).…”

Section: Discussionmentioning

confidence: 56%

Section: Discussionmentioning

confidence: 99%

Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA ₀ Cleavage Site

Diederich

Berhane

Embury-Hyatt

et al. 2015

J Virol

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“…Most notably, the reassortants Hp-H2 poly , Hp-H4 poly , Hp-H8 poly , and Hp-H14 poly displayed a highly pathogenic phenotype as they led to death in all animals within 7 d after oculonasal infection. With an IVPI of 2.37, 2.79, 2.85, or 2.61, these viruses are considerably more virulent than previously reported artificial non-H5/non-H7 HPAIV like an H6 polybasic HACS mutant [IVPI 1.4 (21)] or an H9 HP reassortant [IVPI 1.23 (22)]. The lower clinical score of Hp-H14 poly after oculonasal infection, in contrast to the three other lethal reassortants, suggests that more adaptation would be required.…”

Section: Discussionmentioning

confidence: 73%

“…The lower clinical score of Hp-H14 poly after oculonasal infection, in contrast to the three other lethal reassortants, suggests that more adaptation would be required. As the IVPIs of Hp-H2 poly , Hp-H4 poly , Hp-H8 poly , and Hp-H14 poly are indistinguishable from those of genuine HPAIV, e.g., the strain A/Chicken/Pennsylvania/1370/83 (H5N2) with an IVPI of 2.37 (23) or the parental Hp-Wt with an IVPI of 2.88 (22,24), our data indicate that non-H5/H7 HA with an engineered polybasic HACS can be functionally equivalent to the HA of natural HPAIV.…”

Section: Discussionmentioning

confidence: 99%

Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype

Veits

Weber²,

Stech³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

117

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High-pathogenic avian influenza viruses (HPAIVs) evolve from lowpathogenic precursors specifying the HA serotypes H5 or H7 by acquisition of a polybasic HA cleavage site. As the reason for this serotype restriction has remained unclear, we aimed to distinguish between compatibility of a polybasic cleavage site with H5/H7 HA only and unique predisposition of these two serotypes for insertion mutations. To this end, we introduced a polybasic cleavage site into the HA of several low-pathogenic avian strains with serotypes H1, H2, H3, H4, H6, H8, H10, H11, H14, or H15, and rescued HA reassortants after cotransfection with the genes from either a low-pathogenic H9N2 or high-pathogenic H5N1 strain. Oculonasal inoculation with those reassortants resulted in varying pathogenicity in chicken. Recombinants containing the engineered H2, H4, H8, or H14 in the HPAIV background were lethal and exhibited i.v. pathogenicity indices of 2.79, 2.37, 2.85, and 2.61, respectively, equivalent to naturally occurring H5 or H7 HPAIV. Moreover, the H2, H4, and H8 reassortants were transmitted to some contact chickens. The H2 reassortant gained two mutations in the M2 proton channel gate region, which is affected in some HPAIVs of various origins. Taken together, in the presence of a polybasic HA cleavage site, non-H5/H7 HA can support a highly pathogenic phenotype in the appropriate viral background, indicating requirement for further adaptation. Therefore, the restriction of natural HPAIV to serotypes H5 and H7 is likely a result of their unique predisposition for acquisition of a polybasic HA cleavage site.

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“…Eight plasmids (pHW2000-R66-PB2, pHW2000-R66-PB1, pHW2000-R66-PA, pHW2000-R66-NP, pHW2000-R66-NA, pHW2000-R66-M, pHW2000-R66-NS, and pHW2000-R66-HA), encoding the individual viral RNA segments for influenza A virus A/chicken/Emirates/R66/2002 (H9N2) (R66) (GenBank accession numbers CY076720 to CY076727), were applied to generate recombinant viruses (68,69). To generate a mutant virus, site-directed mutagenesis was performed on the pHW2000-R66-HA plasmid using the QuikChange lightning site-directed mutagenesis kit (Agilent Technologies).…”

Section: Methodsmentioning

confidence: 99%

Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice

Yang

Punyadarsaniya

Lambertz

et al. 2017

J Virol

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The natural reservoir for influenza viruses is waterfowl, and from there they succeeded in crossing the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precisioncut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1 to P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized ␣2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with ␣2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190ϩHA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. In contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs.IMPORTANCE Swine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but have not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by passaging the virus three times in differentiated porcine respiratory epithelial cells. Among the four mutations detected, the two HA mutations were analyzed by generating recombinant viruses. Depending on the infection system used, the mutations differed in their phenotypic expression, e.g., sialic acid binding activity, replication kinetics, plaque size, and pathogenicity in inbred mice. However, none of the mutations affected the ciliary activity which serves as a virulence marker. Thus, early

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H9 avian influenza reassortant with engineered polybasic cleavage site displays a highly pathogenic phenotype in chicken

Cited by 49 publications

References 56 publications

Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA ₀ Cleavage Site

Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA ₀ Cleavage Site

Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype

Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice

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H9 avian influenza reassortant with engineered polybasic cleavage site displays a highly pathogenic phenotype in chicken

Cited by 49 publications

References 56 publications

Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA 0 Cleavage Site

Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA 0 Cleavage Site

Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype

Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice

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Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA ₀ Cleavage Site

Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA ₀ Cleavage Site