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. 2021 Aug 11;13(8):1584.
doi: 10.3390/v13081584.

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

Anastasia Treshchalina  1 , Yulia Postnikova  2 , Elizaveta Boravleva  1 , Alexandra Gambaryan  1 , Alla Belyakova  1 , Aydar Ishmukhametov  1 , Galina Sadykova  3 , Alexey Prilipov  3 , Natalia Lomakina  3
Affiliations

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

Anastasia Treshchalina et al. Viruses. .

Abstract

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.

Keywords: highly pathogenic avian influenza viruses; pathogenicity factors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Arg140 arrangement in HA. Atomic coordinates of H7 HA (1ti8) were used [24]. (A) The protein surface is colored blue. The key amino acids of RBS are colored red. The Arg140 is colored by element. (B) Protein chain of HA near the RBS. The key amino acids of RBS are colored red and marked. The Arg140 and neighboring amino acids are colored by element.
Figure 2
Figure 2
Antibodies against homologous virus measured in ELISA in serum of mice infected with 101 EID50 of R5p. Blue curves show levels of signal for 6 infected mice. Red curves show levels of signal for 2 non-infected mice. The first point in all curves shows signals in control well (column H without virus, see Section 2.13).
Figure 3
Figure 3
Weight dynamics and survival of mice infected with R5p, R6p and the A/mallard/Sweden/91/02 (H7N9) virus. Result of a typical experiment is presented.
Figure 4
Figure 4
The pH dependence of erythrocyte hemolysis in complex with R5p and R6p viruses. A415—absorption at 415 nm, which corresponds to the degree of erythrocyte hemolysis. Result of a typical experiment is presented.
Figure 5
Figure 5
Affinity of peroxidase-labeled fetuin for virions of R5p and R6p strains. The results of testing Fet-HRP on a plate coated with R5p and R6p viruses are presented in Scatchard coordinates. The X-axis is the A450 signal reflecting the amount of bound fetuin, and the Y-axis is the ratio of the A450 signal to the fetuin concentration (expressed in µM of sialic acid) in the solution. Result of a typical experiment is presented.
Figure 6
Figure 6
Inhibition of labeled fetuin binding to R5p and R6p viruses by the receptor analogs. Receptor analogs were added to a solution of labeled fetuin at the indicated concentrations and the level of binding of the label to the virus was measured. Result of a typical experiment is presented.
Figure 7
Figure 7
Different pictures of cells infected with R5p (A) and R6p (B) variants of influenza virus Rostock.
See this image and copyright information in PMC

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