Combined insertion of basic and non-basic amino acids at hemagglutinin cleavage site of highly pathogenic H7N9 virus promotes replication and pathogenicity in chickens and mice

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥ 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

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An avian influenza virus of H10 subtype that is highly pathogenic for chickens, but lacks multiple basic amino acids at the haemagglutinin cleavage site

Avian Pathology ◽

10.1080/03079459608419182 ◽

1996 ◽

Vol 25 (4) ◽

pp. 799-806 ◽

Cited By ~ 38

Author(s):

G. W. Wood ◽

J. Banks ◽

I. Strong ◽

G. Parsons ◽

D. J. Alexander

Keyword(s):

Amino Acids ◽

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Cleavage Site ◽

Highly Pathogenic ◽

Basic Amino Acids

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Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV) Reduced Virus Fitness in Chickens which is Restored by Reassortment with Highly Pathogenic H5N1 AIV

10.20944/preprints202002.0391.v1 ◽

2020 ◽

Author(s):

Marcel Gischke ◽

Reiner Ulrich ◽

Olanrewaju I. Fatola ◽

David Scheibner ◽

Ahmed H. Salaheldin ◽

...

Keyword(s):

Amino Acids ◽

Avian Influenza ◽

Cleavage Site ◽

Influenza Viruses ◽

Viral Fitness ◽

Highly Pathogenic ◽

Basic Amino Acids ◽

The Impact ◽

Virus Fitness

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in the hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥1.2. Only few non-H5/H7 viruses fulfill the criteria of HPAIVs; nevertheless, it remains unknown why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIVs H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Likewise, replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIVs in poultry.

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Molecular Determinants within the Surface Proteins Involved in the Pathogenicity of H5N1 Influenza Viruses in Chickens

Journal of Virology ◽

10.1128/jvi.78.18.9954-9964.2004 ◽

2004 ◽

Vol 78 (18) ◽

pp. 9954-9964 ◽

Cited By ~ 91

Author(s):

Diane J. Hulse ◽

Robert G. Webster ◽

Rupert J. Russell ◽

Daniel R. Perez

Keyword(s):

Amino Acids ◽

Avian Influenza ◽

Cleavage Site ◽

Glycosylation Site ◽

Influenza Viruses ◽

Surface Proteins ◽

Avian Influenza Viruses ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

High Pathogenicity

ABSTRACT Although it is established that the cleavage site and glycosylation patterns in the hemagglutinin (HA) play important roles in determining the pathogenicity of H5 avian influenza viruses, some viruses exist that are not highly pathogenic despite possessing the known characteristics of high pathogenicity (i.e., their HA contains multiple basic amino acids at the cleavage site and has glycosylation patterns similar to that of the highly pathogenic H5 viruses). Currently little is known about the H5N1 viruses that fall into this intermediate category of pathogenicity. We have identified strains of H5N1 avian influenza viruses that have markers typical of high pathogenicity but distinctly differ in their ability to cause disease and death in chickens. By analyzing viruses constructed by reverse-genetic methods and containing recombinant HAs, we established that amino acids 97, 108, 126, 138, 212, and 217 of HA, in addition to those within the cleavage site, affect pathogenicity. Further investigation revealed that an additional glycosylation site within the neuraminidase (NA) protein globular head contributed to the high virulence of the H5N1 virus. Our findings are in agreement with previous observations that suggest that the activities of the HA and NA proteins are functionally linked.

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First genome report and analysis of chicken H7N9 influenza viruses with poly-basic amino acids insertion in the hemagglutinin cleavage site

Scientific Reports ◽

10.1038/s41598-017-10605-6 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 12

Author(s):

Jidang Chen ◽

Jipei Zhang ◽

Wanjun Zhu ◽

Yishan Zhang ◽

Hualong Tan ◽

...

Keyword(s):

Amino Acids ◽

Cleavage Site ◽

Influenza Viruses ◽

Basic Amino Acids ◽

H7n9 Influenza

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SequenceAnalysis of Recent H7 Avian Influenza Viruses Associated with ThreeDifferent Outbreaks in Commercial Poultry in the UnitedStates

Journal of Virology ◽

10.1128/jvi.77.24.13399-13402.2003 ◽

2003 ◽

Vol 77 (24) ◽

pp. 13399-13402 ◽

Cited By ~ 72

Author(s):

Erica Spackman ◽

Dennis A. Senne ◽

Sherrill Davison ◽

David L. Suarez

Keyword(s):

Amino Acids ◽

Avian Influenza ◽

Cleavage Site ◽

Influenza Viruses ◽

Avian Influenza Viruses ◽

Receptor Binding Site ◽

Highly Pathogenic ◽

Genetic Features ◽

Commercial Poultry ◽

Live Bird

ABSTRACT The hemagglutinin (HA) and neuraminidase (NA) genes of H7 avian influenza virus (AIV) isolated between 1994 and 2002 from live-bird markets (LBMs) in the northeastern United States and from three outbreaks in commercial poultry have been characterized. Phylogenetic analysis of the HA and NA genes demonstrates that the isolates from commercial poultry were closely related to the viruses circulating in the LBMs. Also, since 1994, two distinguishing genetic features have appeared in this AIV lineage: a deletion of 17 amino acids in the NA protein stalk region and a deletion of 8 amino acids in the HA1 protein which is putatively in part of the receptor binding site. Furthermore, analysis of the HA cleavage site amino acid sequence, a marker for pathogenicity in chickens and turkeys, shows a progression toward a cleavage site sequence that fulfills the molecular criteria for highly pathogenic AIV.

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Structural Requirements in the Hemagglutinin Cleavage Site-Coding RNA Region for the Generation of Highly Pathogenic Avian Influenza Virus

Pathogens ◽

10.3390/pathogens10121597 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1597

Author(s):

Yurie Kida ◽

Kosuke Okuya ◽

Takeshi Saito ◽

Junya Yamagishi ◽

Aiko Ohnuma ◽

...

Keyword(s):

Amino Acids ◽

Cleavage Site ◽

Highly Pathogenic Avian Influenza ◽

Loop Structure ◽

Highly Pathogenic ◽

Stem Loop ◽

Pathogenic Avian Influenza ◽

Ha Gene ◽

Stem Loop Structure ◽

Nucleotide Insertions

Highly pathogenic avian influenza viruses (HPAIVs) with H5 and H7 hemagglutinin (HA) subtypes are derived from their low pathogenic counterparts following the acquisition of multiple basic amino acids in their HA cleavage site. It has been suggested that consecutive adenine residues and a stem-loop structure in the viral RNA region that encodes the cleavage site are essential for the acquisition of the polybasic cleavage site. By using a reporter assay to detect non-templated nucleotide insertions, we found that insertions more frequently occurred in the RNA region (29 nucleotide-length) encoding the cleavage site of an H5 HA gene that was predicted to have a stem-loop structure containing consecutive adenines than in a mutated corresponding RNA region that had a disrupted loop structure with fewer adenines. In virus particles generated by using reverse genetics, nucleotide insertions that created additional codons for basic amino acids were found in the RNA region encoding the cleavage site of an H5 HA gene but not in the mutated RNA region. We confirmed the presence of virus clones with the ability to replicate without trypsin in a plaque assay and to cause lethal infection in chicks. These results demonstrate that the stem-loop structure containing consecutive adenines in HA genes is a key molecular determinant for the emergence of H5 HPAIVs.

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Insertions of codons encoding basic amino acids in H7 hemagglutinins of influenza A viruses occur by recombination with RNA at hotspots near snoRNA binding sites

10.1101/2020.09.21.303073 ◽

2020 ◽

Author(s):

Alexander P. Gultyaev ◽

Monique I. Spronken ◽

Mathis Funk ◽

Ron A.M. Fouchier ◽

Mathilde Richard

Keyword(s):

Amino Acids ◽

Cleavage Site ◽

Influenza A ◽

Rna Recombination ◽

Influenza A Viruses ◽

Rna Sequences ◽

Rna Molecules ◽

Basic Amino Acids ◽

Molecular Determinant ◽

Ha Protein

ABSTRACTThe presence of multiple basic amino acids in the protease cleavage site of the hemagglutinin (HA) protein is the main molecular determinant of virulence of highly pathogenic avian influenza (HPAI) viruses. Recombination of HA RNA with other RNA molecules of host or virus origin is a dominant mechanism of multi basic cleavage site (MBCS) acquisition for H7 subtype HA. Using alignments of HA RNA sequences from documented cases of MBCS insertion due to recombination, we show that such recombination with host RNAs is most likely to occur at particular hotspots in ribosomal RNAs (rRNAs), transfer RNAs (tRNAs) and viral RNAs. The locations of these hotspots in highly abundant RNAs indicate that RNA recombination is facilitated by the binding of small nucleolar RNA (snoRNA) near the recombination points.

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Multiple basic amino acids in the cleavage site of H7N9 hemagglutinin contribute to high virulence in mice

Journal of Thoracic Disease ◽

10.21037/jtd-21-226 ◽

2021 ◽

Vol 0 (0) ◽

pp. 0-0

Author(s):

Wenjun Song ◽

Xiaofeng Huang ◽

Wenda Guan ◽

Pin Chen ◽

Pui Wang ◽

...

Keyword(s):

Amino Acids ◽

Cleavage Site ◽

Basic Amino Acids ◽

High Virulence

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Non-basic amino acids in the hemagglutinin proteolytic cleavage site of a European H9N2 avian influenza virus modulate virulence in turkeys

Scientific Reports ◽

10.1038/s41598-020-78210-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Claudia Blaurock ◽

David Scheibner ◽

Maria Landmann ◽

Melina Vallbracht ◽

Reiner Ulrich ◽

...

Keyword(s):

Amino Acids ◽

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Cleavage Site ◽

Mammalian Cells ◽

Proteolytic Cleavage ◽

Brain Cells ◽

H9n2 Avian Influenza Virus ◽

Basic Amino Acids

AbstractH9N2 avian influenza virus (AIV) is the most widespread low pathogenic (LP) AIV in poultry and poses a serious zoonotic risk. Vaccination is used extensively to mitigate the economic impact of the virus. However, mutations were acquired after long-term circulation of H9N2 virus in poultry, particularly in the hemagglutinin (HA) proteolytic cleavage site (CS), a main virulence determinant of AIV. Compared to chickens, little is known about the genetic determinants for adaptation of H9N2 AIV to turkeys. Here, we describe 36 different CS motifs in Eurasian H9N2 viruses identified from 1966 to 2019. The European H9N2 viruses specify unique HACS with particular polymorphism by insertion of non-basic amino acids at position 319. Recombinant viruses carrying single HACS mutations resembling field viruses were constructed (designated G319, A319, N319, S319, D319 and K319). Several viruses replicated to significantly higher titers in turkey cells than in chicken cells. Serine proteases were more efficient than trypsin to support multicycle replication in mammalian cells. Mutations affected cell-to-cell spread and pH-dependent HA fusion activity. In contrast to chickens, mutations in the HACS modulated clinical signs in inoculated and co-housed turkeys. G319 exhibited the lowest virulence, however, it replicated to significantly higher titers in contact-turkeys and in vitro. Interestingly, H9N2 viruses, particularly G319, replicated in brain cells of turkeys and to a lesser extent in mammalian brain cells independent of trypsin. Therefore, the silent circulation of potentially zoonotic H9N2 viruses in poultry should be monitored carefully. These results are important for understanding the adaptation of H9N2 in poultry and replication in mammalian cells.

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