scholarly journals Substitution Arg140Gly in Hemagglutinin Reduced the Virulence of Highly Pathogenic Avian Influenza Virus H7N1

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1584
Author(s):  
Anastasia Treshchalina ◽  
Yulia Postnikova ◽  
Elizaveta Boravleva ◽  
Alexandra Gambaryan ◽  
Alla Belyakova ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Receptor Binding ◽  
Genome Stability ◽  
Laboratory Strain ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Head Part ◽  
Receptor Binding Site ◽  
Highly Pathogenic

The H7 subtype of avian influenza viruses (AIV) stands out among other AIV. The H7 viruses circulate in ducks, poultry and equines and have repeatedly caused outbreaks of disease in humans. The laboratory strain A/chicken/Rostock/R0p/1934 (H7N1) (R0p), which was previously derived from the highly pathogenic strain A/FPV/Rostock/1934 (H7N1), was studied in this work to ascertain its biological property, genome stability and virulent changing mechanism. Several virus variants were obtained by serial passages in the chicken lungs. After 10 passages of this virus through the chicken lungs we obtained a much more pathogenic variant than the starting R0p. The study of intermediate passages showed a sharp increase in pathogenicity between the fifth and sixth passage. By cloning these variants, a pair of strains (R5p and R6p) was obtained, and the complete genomes of these strains were sequenced. Single amino acid substitution was revealed, namely reversion Gly140Arg in HA1. This amino acid is located at the head part of the hemagglutinin, adjacent to the receptor-binding site. In addition to the increased pathogenicity in chicken and mice, R6p differs from R5p in the shape of foci in cell culture and an increased affinity for a negatively charged receptor analogue, while maintaining a pattern of receptor-binding specificity and the pH of conformational change of HA.

scholarly journals Substitution Arg140Gly in HA/H7 Reduced the Virulence Highly Pathogenic Avian Influenza Virus H7N1

2021 ◽  
Author(s):  
Anastasia Treshchalina ◽  
Yulia Postnikova ◽  
Elizaveta Boravleva ◽  
Alexandra Gambaryan ◽  
Alla Belyakova ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Genome Stability ◽  
Laboratory Strain ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Head Part ◽  
Receptor Binding Site ◽  
Ph Optimum ◽  
History Of

The H7 subtype of avian influenza viruses (AIV) stands out among other AIV. The H7 viruses cir-culate in ducks, poultry, equine and have repeatedly caused outbreaks of disease in humans. In or-der to study the pathogenicity factors of H7N1 viruses, several variants were obtained, starting with laboratory strain, with a history of 12 passages through chicken embryos. This strain, A/chicken/Rostock/R0p/1934(H7N1) (R0p) had only 3 substitution in HA relatively A/Chicken/Rostock/45/34(H7N1), substitution Arg140Gly among them. 10 variants of this strain was obtained and studied to ascertain its biological property, genome stability and factors of patho-genicity. Strain R0p had decreased virulence for chicken, comparing with described in literature virulence of A/FPV Rostock/34 and A/chicken/Rostock/34 viruses. After 10 passages through the chicken lungs variant was obtained much more pathogenic than the starting R0p. The study of in-termediate passages through the chicken lungs showed that the jump in pathogenicity had occurred sharply between the fifth and sixth passage. By cloning these variants, a pair of strains (R5p and R6p) were obtained, and the complete genomes of these strains were sequenced. Single amino acid substitution was revealed, namely reversion Gly140Arg in HA1. This amino acid is located at the head part of the hemagglutinin, adjacent to the receptor-binding site. In addition to the increased pathogenicity for chicken and mice, R6p differs from R5p in the pattern of foci in cell culture and an increased affinity for a negatively charged receptor analogue, while maintaining a pattern of recep-tor binding specificity and the pH optimum of the HA conformational change.

scholarly journals Substitution Arg140Gly in HA/H7 attenuated highly pathogenic avian influenza virus FPV/Rostock/34 (H7N1)

2021 ◽  
Author(s):  
Yulia Postnikova ◽  
Anastasia Treshchalina ◽  
Elizaveta Boravleva ◽  
Alexandra Gambaryan ◽  
Aydar Ishmukhametov ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Head Part ◽  
Receptor Binding Site ◽  
Original Laboratory ◽  
Pathogenic Avian Influenza Virus ◽  
H7n1 Virus

The H7 subtype of avian influenza viruses (AIV) stands out among other AIV. H7 viruses circulate in ducks, poultry, equine and have repeatedly caused outbreaks of disease in humans. In order to study the pathogenicity factors of H7N1 viruses, several laboratory variants of the A/FPV/Rostock/34 (H7N1) strain were obtained by passages in the chicken lungs. After 10 such passages, a variant was obtained that differed from the parent virus by amino acid substitutions Val109Phe in PB2, Gln621Lys in PB1, Thr32Ala and Leu586Phe in PA Gly140Arg in HA1 and Ala101Thr in HA2 (numbering by H3), Ser82Arg in M2, Arg118Lys and Met124Arg in NS1. No differences were found in proteins NA, NP, M1 and NS2. The resulting variant was hundreds of times more pathogenic for chickens than the original laboratory variant of the virus. The study of intermediate passages showed that the jump in pathogenicity occurs sharply between the fifth and sixth passage through the chicken lungs. By cloning these variants, a pair of strains (R5p and R6p) were obtained, and the complete genomes of these strains were sequenced. Single amino acid substitution was revealed, namely Gly140Arg in HA1. It is important to emphasize that this substitution is a reversion, since Arg is located in position 140 HA1 of original the A/FPV/Rostock/34 (H7N1) virus (GenBank). This amino acid is located at the head part of the hemagglutinin, adjacent to the receptor-binding site. In addition to the increased pathogenicity, R6p differs from R5p by an increased affinity for a negatively charged receptor analogue, an increased affinity for MDCK cells, while maintaining a receptor specificity profile.

scholarly journals Phenotypic Effects of Substitutions within the Receptor Binding Site of Highly Pathogenic Avian Influenza H5N1 Virus Observed during Human Infection

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Dirk Eggink ◽  
Monique Spronken ◽  
Roosmarijn van der Woude ◽  
Jocynthe Buzink ◽  
Frederik Broszeit ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Binding Site ◽  
Receptor Binding ◽  
Human Infection ◽  
Influenza Viruses ◽  
Receptor Specificity ◽  
Receptor Binding Site ◽  
Human Type ◽  
Human Infections

ABSTRACT Highly pathogenic avian influenza (HPAI) viruses are enzootic in wild birds and poultry and continue to cause human infections with high mortality. To date, more than 850 confirmed human cases of H5N1 virus infection have been reported, of which ∼60% were fatal. Global concern persists that these or similar avian influenza viruses will evolve into viruses that can transmit efficiently between humans, causing a severe influenza pandemic. It was shown previously that a change in receptor specificity is a hallmark for adaptation to humans and evolution toward a transmittable virus. Substantial genetic diversity was detected within the receptor binding site of hemagglutinin of HPAI A/H5N1 viruses, evolved during human infection, as detected by next-generation sequencing. Here, we investigated the functional impact of substitutions that were detected during these human infections. Upon rescue of 21 mutant viruses, most substitutions in the receptor binding site (RBS) resulted in viable virus, but virus replication, entry, and stability were often impeded. None of the tested substitutions individually resulted in a clear switch in receptor preference as measured with modified red blood cells and glycan arrays. Although several combinations of the substitutions can lead to human-type receptor specificity, accumulation of multiple amino acid substitutions within a single hemagglutinin during human infection is rare, thus reducing the risk of virus adaptation to humans. IMPORTANCE H5 viruses continue to be a threat for public health. Because these viruses are immunologically novel to humans, they could spark a pandemic when adapted to transmit between humans. Avian influenza viruses need several adaptive mutations to bind to human-type receptors, increase hemagglutinin (HA) stability, and replicate in human cells. However, knowledge on adaptive mutations during human infections is limited. A previous study showed substantial diversity within the receptor binding site of H5N1 during human infection. We therefore analyzed the observed amino acid changes phenotypically in a diverse set of assays, including virus replication, stability, and receptor specificity. None of the tested substitutions resulted in a clear step toward a human-adapted virus capable of aerosol transmission. It is notable that acquiring human-type receptor specificity needs multiple amino acid mutations, and that variability at key position 226 is not tolerated, reducing the risk of them being acquired naturally.

scholarly journals The NS1 Gene Contributes to the Virulence of H5N1 Avian Influenza Viruses

2006 ◽  
Vol 80 (22) ◽  
pp. 11115-11123 ◽  
Author(s):  
Zejun Li ◽  
Yongping Jiang ◽  
Peirong Jiao ◽  
Aiqin Wang ◽  
Fengju Zhao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Recombinant Virus ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Ns1 Protein ◽  
Highly Pathogenic ◽  
H5n1 Influenza ◽  
Ns1 Gene

ABSTRACT In the present study, we explored the genetic basis underlying the virulence and host range of two H5N1 influenza viruses in chickens. A/goose/Guangdong/1/96 (GS/GD/1/96) is a highly pathogenic virus for chickens, whereas A/goose/Guangdong/2/96 (GS/GD/2/96) is unable to replicate in chickens. These two H5N1 viruses differ in sequence by only five amino acids mapping to the PA, NP, M1, and NS1 genes. We used reverse genetics to create four single-gene recombinants that contained one of the sequence-differing genes from nonpathogenic GS/GD/2/96 and the remaining seven gene segments from highly pathogenic GS/GD/1/96. We determined that the NS1 gene of GS/GD/2/96 inhibited the replication of GS/GD/1/96 in chickens, while the substitution of the PA, NP, or M gene did not change the highly pathogenic properties of GS/GD/1/96. Conversely, of the recombinant viruses generated in the GS/GD/2/96 background, only the virus containing the NS1 gene of GS/GD/1/96 was able to replicate and cause disease and death in chickens. The single-amino-acid difference in the sequence of these two NS1 genes resides at position 149. We demonstrate that a recombinant virus expressing the GS/GD/1/96 NS1 protein with Ala149 is able to antagonize the induction of interferon protein levels in chicken embryo fibroblasts (CEFs), but a recombinant virus carrying a Val149 substitution is not capable of the same effect. These results indicate that the NS1 gene is critical for the pathogenicity of avian influenza virus in chickens and that the amino acid residue Ala149 correlates with the ability of these viruses to antagonize interferon induction in CEFs.

scholarly journals A Single Amino Acid Substitution at Residue 218 of Hemagglutinin Improves the Growth of Influenza A(H7N9) Candidate Vaccine Viruses

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Xing Li ◽  
Yamei Gao ◽  
Zhiping Ye
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Receptor Binding ◽  
Influenza A ◽  
Influenza Pandemic ◽  
Single Amino Acid ◽  
Virus Release ◽  
Highly Pathogenic ◽  
H7n9 Influenza ◽  
Human Infections

ABSTRACTThe potential avian influenza pandemic remains a threat to public health, as the avian-origin influenza A(H7N9) virus has caused more than 1,560 laboratory-confirmed human infections since 2013, with nearly 40% mortality. Development of low-pathogenic candidate vaccine viruses (CVVs) for vaccine production is essential for pandemic preparedness. However, the suboptimal growth of CVVs in mammalian cells and chicken eggs is often a challenge. By introducing a single adaptive substitution, G218E, into the hemagglutinin (HA), we generated reassortant A(H7N9)-G218E CVVs that were characterized by significantly enhanced growth in both cells and eggs. These G218E CVVs retained the original antigenicity, as determined by a hemagglutination inhibition assay, and effectively protected ferrets from lethal challenge with the highly pathogenic parental virus. We found that the suboptimal replication of the parental H7 CVVs was associated with impeded progeny virus release as a result of strong HA receptor binding relative to weak neuraminidase (NA) cleavage of receptors. In contrast, the G218E-mediated growth improvement was attributed to relatively balanced HA and NA functions, resulted from reduced HA binding to both human- and avian-type receptors, and thus facilitated NA-mediated virus release. Our findings revealed that a single amino acid mutation at residue 218 of the HA improved the growth of A(H7N9) influenza virus by balancing HA and NA functions, shedding light on an alternative approach for optimizing certain influenza CVVs.IMPORTANCEThe circulating avian influenza A(H7N9) has caused recurrent epidemic waves with high mortality in China since 2013, in which the alarming fifth wave crossing 2016 and 2017 was highlighted by a large number of human infections and the emergence of highly pathogenic avian influenza (HPAI) A(H7N9) strains in human cases. We generated low-pathogenic reassortant CVVs derived from the emerging A(H7N9) with improved virus replication and protein yield in both MDCK cells and eggs by introducing a single substitution, G218E, into HA, which was associated with reducing HA receptor binding and subsequently balancing HA-NA functions. Thein vitroandin vivoexperiments demonstrated comparable antigenicity of the G218E CVVs with that of their wild-type (WT) counterparts, and both the WT and the G218E CVVs fully protected ferrets from parental HPAI virus challenge. With high yield traits and the anticipated antigenicity, the G218E CVVs should benefit preparedness against the threat of an A(H7N9) influenza pandemic.

scholarly journals Amino acid substitutions in the H5N1 avian influenza haemagglutinin alter pH of fusion and receptor binding to promote a highly pathogenic phenotype in chickens

2021 ◽  
Vol 102 (11) ◽  
Author(s):  
Joshua E. Sealy ◽  
Wendy A. Howard ◽  
Eleonora Molesti ◽  
Munir Iqbal ◽  
Nigel J. Temperton ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Binding Site ◽  
Receptor Binding ◽  
Viral Fitness ◽  
Amino Acid Substitutions ◽  
Receptor Binding Site ◽  
Highly Pathogenic ◽  
Ha Gene ◽  
H5n1 Avian Influenza

Highly pathogenic H5N1 avian influenza viruses cause devastating outbreaks in farmed poultry with serious consequences for animal welfare and economic losses. Zoonotic infection of humans through close contact with H5N1 infected birds is often severe and fatal. England experienced an outbreak of H5N1 in turkeys in 1991 that led to thousands of farmed bird mortalities. Isolation of clonal populations of one such virus from this outbreak uncovered amino acid differences in the virus haemagglutinin (HA) gene whereby the different genotypes could be associated with distinct pathogenic outcomes in chickens; both low pathogenic (LP) and high pathogenic (HP) phenotypes could be observed despite all containing a multi-basic cleavage site (MBCS) in the HA gene. Using reverse genetics, three amino acid substitutions in HA were examined for their ability to affect pathogenesis in the chicken. Restoration of amino acid polymorphisms close to the receptor binding site that are commonly found in H5 viruses only partially improved viral fitness in vitro and in vivo. A third novel substitution in the fusion peptide, HA2G4R, enabled the HP phenotype. HA2G4R decreased the pH stability of HA and increased the pH of HA fusion. The substitutions close to the receptor binding site optimised receptor binding while modulating the pH of HA fusion. Importantly, this study revealed pathogenic determinants beyond the MBCS.

scholarly journals H5N2 Avian Influenza Outbreak in Texas in 2004: the First Highly Pathogenic Strain in the United States in 20 Years?

2005 ◽  
Vol 79 (17) ◽  
pp. 11412-11421 ◽  
Author(s):  
Chang-Won Lee ◽  
David E. Swayne ◽  
Jose A. Linares ◽  
Dennis A. Senne ◽  
David L. Suarez
Keyword(s):  
United States ◽  
Amino Acid ◽  
Avian Influenza ◽  
Cleavage Site ◽  
Basic Amino Acid ◽  
The United States ◽  
Influenza Viruses ◽  
Single Point Mutation ◽  
Highly Pathogenic

ABSTRACT In early 2004, an H5N2 avian influenza virus (AIV) that met the molecular criteria for classification as a highly pathogenic AIV was isolated from chickens in the state of Texas in the United States. However, clinical manifestations in the affected flock were consistent with avian influenza caused by a low-pathogenicity AIV and the representative virus (A/chicken/Texas/298313/04 [TX/04]) was not virulent for experimentally inoculated chickens. The hemagglutinin (HA) gene of the TX/04 isolate was similar in sequence to A/chicken/Texas/167280-4/02 (TX/02), a low-pathogenicity AIV isolate recovered from chickens in Texas in 2002. However, the TX/04 isolate had one additional basic amino acid at the HA cleavage site, which could be attributed to a single point mutation. The TX/04 isolate was similar in sequence to TX/02 isolate in several internal genes (NP, M, and NS), but some genes (PA, PB1, and PB2) had sequence of a clearly different origin. The TX/04 isolate also had a stalk deletion in the NA gene, characteristic of a chicken-adapted AIV. By analyzing viruses constructed by in vitro mutagenesis followed by reverse genetics, we found that the pathogenicity of the TX/04 virus could be increased in vitro and in vivo by the insertion of an additional basic amino acid at the HA cleavage site and not by the loss of a glycosylation site near the cleavage site. Our study provides the genetic and biologic characteristics of the TX/04 isolate, which highlight the complexity of the polygenic nature of the virulence of influenza viruses.

scholarly journals A Single-Amino-Acid Substitution in the NS1 Protein Changes the Pathogenicity of H5N1 Avian Influenza Viruses in Mice

2007 ◽  
Vol 82 (3) ◽  
pp. 1146-1154 ◽  
Author(s):  
Peirong Jiao ◽  
Guobin Tian ◽  
Yanbing Li ◽  
Guohua Deng ◽  
Yongping Jiang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Host Cell ◽  
Amino Acid Substitution ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Ns1 Protein ◽  
Avian Influenza Viruses ◽  
H5n1 Influenza ◽  
H5n1 Avian Influenza

ABSTRACT In this study, we explored the molecular basis determining the virulence of H5N1 avian influenza viruses in mammalian hosts by comparing two viruses, A/Duck/Guangxi/12/03 (DK/12) and A/Duck/Guangxi/27/03 (DK/27), which are genetically similar but differ in their pathogenicities in mice. To assess the genetic basis for this difference in virulence, we used reverse genetics to generate a series of reassortants and mutants of these two viruses. We found that a single-amino-acid substitution of serine for proline at position 42 (P42S) in the NS1 protein dramatically increased the virulence of the DK/12 virus in mice, whereas the substitution of proline for serine at the same position (S42P) completely attenuated the DK/27 virus. We further demonstrated that the amino acid S42 of NS1 is critical for the H5N1 influenza virus to antagonize host cell interferon induction and for the NS1 protein to prevent the double-stranded RNA-mediated activation of the NF-κB pathway and the IRF-3 pathway. Our results indicate that the NS1 protein is critical for the pathogenicity of H5N1 influenza viruses in mammalian hosts and that the amino acid S42 of NS1 plays a key role in undermining the antiviral immune response of the host cell.

scholarly journals Identification of Stabilizing Mutations in an H5 Hemagglutinin Influenza Virus Protein

2015 ◽  
Vol 90 (6) ◽  
pp. 2981-2992 ◽  
Author(s):  
Anthony Hanson ◽  
Masaki Imai ◽  
Masato Hatta ◽  
Ryan McBride ◽  
Hirotaka Imai ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Conformational Change ◽  
Receptor Binding ◽  
Heat Stability ◽  
Influenza Viruses ◽  
Virus Infectivity ◽  
Highly Pathogenic ◽  
Human Type ◽  
Droplet Transmission

ABSTRACTHighly pathogenic avian influenza viruses of the H5N1 subtype continue to circulate in poultry in Asia, Africa, and the Middle East. Recently, outbreaks of novel reassortant H5 viruses have also occurred in North America. Although the number of human infections with highly pathogenic H5N1 influenza viruses continues to rise, these viruses remain unable to efficiently transmit between humans. However, we and others have identified H5 viruses capable of respiratory droplet transmission in ferrets. Two experimentally introduced mutations in the viral hemagglutinin (HA) receptor-binding domain conferred binding to human-type receptors but reduced HA stability. Compensatory mutations in HA (acquired during virus replication in ferrets) were essential to restore HA stability. These stabilizing mutations in HA also affected the pH at which HA undergoes an irreversible switch to its fusogenic form in host endosomes, a crucial step for virus infectivity. To identify additional stabilizing mutations in an H5 HA, we subjected a virus library possessing random mutations in the ectodomain of an H5 HA (altered to bind human-type receptors) to three rounds of treatment at 50°C. We isolated several mutants that maintained their human-type receptor-binding preference but acquired an appreciable increase in heat stability and underwent membrane fusion at a lower pH; collectively, these properties may aid H5 virus respiratory droplet transmission in mammals.IMPORTANCEWe have identified mutations in HA that increase its heat stability and affect the pH that triggers an irreversible conformational change (a prerequisite for virus infectivity). These mutations were identified in the genetic background of an H5 HA protein that was mutated to bind to human cells. The ability to bind to human-type receptors, together with physical stability and an altered pH threshold for HA conformational change, may facilitate avian influenza virus transmission via respiratory droplets in mammals.

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