Understanding the Variability of Certain Biological Properties of H1N1pdm09 Influenza Viruses

Abstract

The influenza virus continually evolves because of the high mutation rate, resulting in dramatic changes in its pathogenicity and other biological properties. This study aimed to evaluate the evolution of certain essential properties, understand the connections between them, and find the molecular basis for the manifestation of these properties. To that end, 21 A(H1N1)pdm09 influenza viruses were tested for their pathogenicity and toxicity in a mouse model with a ts/non-ts phenotype manifestation and HA thermal stability. The results demonstrated that, for a strain to have high pathogenicity, it must express a toxic effect, have a non-ts phenotype, and have a thermally stable HA. The ancestor A/California/07/2009 (H1N1)pdm influenza virus expressed the non-ts phenotype, after which the cycling trend of the ts/non-ts phenotype was observed in new strains of A(H1N1)pdm09 influenza viruses, indicating that the ratio of the ts phenotype will increase in the coming years. Of the 21 tested viruses, A/South Africa/3626/2013 had the high pathogenicity in the mouse model. Sequence alignment analysis showed that this virus has three unique mutations in the polymerase complex, two of which are in the PB2 gene and one that is in the PB1 gene. Further study of these mutations might explain the distinguishing pathogenicity.

Keywords: A(H1N1)pdm09 influenza viruses; evolution; polymerase complex; temperature sensitivity of reproduction; thermal stability of the hemagglutinin; viral pathogenicity; viral toxicity.

Figure 1

Four possible combinations of toxicity and pathogenicity of influenza virus for mice. Virus 1—A/California/07/2009; virus 2—A/Bolivia/559/2013; virus 3—A/Mississippi/10/2013; virus 4—A/New Hampshire/04/2013; virus 5—A/South Africa/3626/2013; virus 6—A/Florida/62/2014; virus 7—A/Laos/1187/2014; virus 8—A/New York/61/2015; virus 9—A/Slovenia/2903/2015; virus 10—A/Bangladesh/3002/2015; virus 11—A/Newcastle/67/2017; virus 12—A/South Australia/272/2017; virus 13—A/New Jersey/13/2018; virus 14—A/Darwin/123/2018; virus 15—A/Brisbane/02/2018; virus 16—A/lowa/59/2018; virus 17—A/lowa/12/2019; virus 18—A/Victoria/2570/2019; virus 19—A/Guangdong-Maonan/SWL1536/2019; virus 20—A/Arkansas/08/2020; virus 21—A/Indiana/02/2020. X-axis—days post-infection. Y-axis—lethality, %. (a) the virus is toxic but not pathogenic for mice; (b) the virus is toxic and pathogenic for mice; (c) the virus is non-toxic and non-pathogenic for mice; (d) the virus is non-toxic but pathogenic for mice. The fourth variant is possible only theoretically. So far, we have not encountered strains with a similar combination of features. As for the other three variants, all the viruses studied were distributed as follows. Group (iii) includes five viruses: A/California/7/2009, A/Slovenia/2903/2015, A/New Jersey/13/2018, A/Darwin/123/2018, and A/Indiana/02/20. Group (ii) included six viruses: A/South Africa/3626/2013, A/Bolivia/559/2013, A/New Hampshire/04/2013, A/Laos/1187/2014, A/Florida/62/2014 and A/Bangladesh/3002/2015 and the first group (i) which has most of the viruses (consisted of ten viruses): A/Mississippi/10/2013, A/New York/61/2015, A/South Australia/272/2017, A/Newcastle/67/2017, A/Iowa/59/2018, A/Brisbane/02/2018, A/Victoria/2570/2019, A/Guangdong-Maonan/SWL1536/2019, A/Iowa/12/2019 and A/Arkansas/08/2020.

Figure 1

Four possible combinations of toxicity and pathogenicity of influenza virus for mice. Virus 1—A/California/07/2009; virus 2—A/Bolivia/559/2013; virus 3—A/Mississippi/10/2013; virus 4—A/New Hampshire/04/2013; virus 5—A/South Africa/3626/2013; virus 6—A/Florida/62/2014; virus 7—A/Laos/1187/2014; virus 8—A/New York/61/2015; virus 9—A/Slovenia/2903/2015; virus 10—A/Bangladesh/3002/2015; virus 11—A/Newcastle/67/2017; virus 12—A/South Australia/272/2017; virus 13—A/New Jersey/13/2018; virus 14—A/Darwin/123/2018; virus 15—A/Brisbane/02/2018; virus 16—A/lowa/59/2018; virus 17—A/lowa/12/2019; virus 18—A/Victoria/2570/2019; virus 19—A/Guangdong-Maonan/SWL1536/2019; virus 20—A/Arkansas/08/2020; virus 21—A/Indiana/02/2020. X-axis—days post-infection. Y-axis—lethality, %. (a) the virus is toxic but not pathogenic for mice; (b) the virus is toxic and pathogenic for mice; (c) the virus is non-toxic and non-pathogenic for mice; (d) the virus is non-toxic but pathogenic for mice. The fourth variant is possible only theoretically. So far, we have not encountered strains with a similar combination of features. As for the other three variants, all the viruses studied were distributed as follows. Group (iii) includes five viruses: A/California/7/2009, A/Slovenia/2903/2015, A/New Jersey/13/2018, A/Darwin/123/2018, and A/Indiana/02/20. Group (ii) included six viruses: A/South Africa/3626/2013, A/Bolivia/559/2013, A/New Hampshire/04/2013, A/Laos/1187/2014, A/Florida/62/2014 and A/Bangladesh/3002/2015 and the first group (i) which has most of the viruses (consisted of ten viruses): A/Mississippi/10/2013, A/New York/61/2015, A/South Australia/272/2017, A/Newcastle/67/2017, A/Iowa/59/2018, A/Brisbane/02/2018, A/Victoria/2570/2019, A/Guangdong-Maonan/SWL1536/2019, A/Iowa/12/2019 and A/Arkansas/08/2020.

Figure 2

Sensitivity of reproduction of A(H1N1)pdm09 influenza viruses to the elevated temperature of 40 °C. Black bars—relative percentage titer at 40 °C compare to infectivity at 32 °C. triangles—pathogenicity for mice. Square—toxicity for mice. Non–ts control 1—A/PR/8/1934; ts control 1—A/Florida/3/2006; virus 1—A/California/07/2009; virus 2—A/Bolivia/559/2013; virus 3—A/Mississippi/10/2013; virus 4—A/New Hampshire/04/2013; virus 5—A/South Africa/3626/2013; virus 6—A/Florida/62/2014; virus 7—A/Laos/1187/2014; virus 8—A/New York/61/2015; virus 9—A/Slovenia/2903/2015; virus 10—A/Bangladesh/3002/2015; virus 11—A/Newcastle/67/2017; virus 12—A/South Australia/272/2017; virus 13—A/New Jersey/13/2018; virus 14—A/Darwin/123/2018; virus 15—A/Brisbane/02/2018; virus 16—A/lowa/59/2018; virus 17—A/lowa/12/2019; virus 18—A/Victoria/2570/2019; virus 19—A/Guangdong-Maonan/SWL1536/2019; virus 20—A/Arkansas/08/2020; virus 21—A/Indiana/02/2020; ts control 2—A/Solomon Islands/3/2006; non–ts control 2—A/New Caledonia/20/1999. The red square indicates a strain of high toxicity. The red triangle indicates strain of high pathogenicity. The pale blue square indicates a strain of low or no toxicity. The pale blue triangle indicates strain of low or no pathogenicity.

Figure 3

Comparison of the dynamic of losses of thermal stability of the hemagglutinin of A(H1N1)pdm09 viruses circulating in 2009–2020. HA activity temperature threshold is (a) 60–65 °C (high thermal stability); (b) 54–58 °C (low thermal stability). RT—room temperature. X-axis—temperature (°C). Y-axis—log2 HA titer. Virus 1—A/California/07/2009; virus 2—A/Bolivia/559/2013; virus 3—A/Mississippi/10/2013; virus 4—A/New Hampshire/04/2013; virus 5—A/South Africa/3626/2013; virus 6—A/Florida/62/2014; virus 7—A/Laos/1187/2014; virus 8—A/New York/61/2015; virus 9—A/Slovenia/2903/2015; virus 10—A/Bangladesh/3002/2015; virus 11—A/Newcastle/67/2017; virus 12—A/South Australia/272/2017; virus 13—A/New Jersey/13/2018; virus 14—A/Darwin/123/2018; virus 15—A/Brisbane/02/2018; virus 16—A/lowa/59/2018; virus 17—A/lowa/12/2019; virus 18—A/Victoria/2570/2019; virus 19—A/Guangdong-Maonan/SWL1536/2019; virus 20—A/Arkansas/08/2020; virus 21—A/Indiana/02/2020. The red square indicates a strain of high toxicity. The red triangle indicates strain of high pathogenicity. The pale blue square indicates a strain of low or no toxicity. The pale blue triangle indicates strain of low or no pathogenicity.

Figure 4

Phylogenetic tree of HA genes of A(H1N1)pdm09 influenza viruses used in this study. The red square indicates a strain of high toxicity. The red triangle indicates a strain of high pathogenicity. The pale blue square indicates a strain of low or no toxicity. The pale blue triangle indicates a strain of low or no pathogenicity.

Figure 5

3D structure of the polymerase complex of A/South Africa/3626/2013 virus. (a) the PB1 protein contains the unique substitution (Gln-687-Arg), (b): the PB2 protein contains two unique substitutions (Asn-102-Thr, Glu-358-Glu/Lys), (c) the PA protein does not contain unique mutations, (d) Structure of the entire polymerase complex with the three substitutions. UCSF Chimera 1.15, was used to build the 3D structure.

Figure 6

Sanger sequencing and pyrosequencing of A/South Africa/3626/2013 virus used for mice infection (egg passage E4) (a): Sanger sequencing of PB2 gene fragment, (b): Detection of the nucleotide sequence in PB2 fragment 1064–1079 of A/South Africa/3626/2013 virus used for mice infection (egg passage E4) by pyrosequencing. The peak highness in G position (b, long arrow) is half-height, peak A (b, short arrow) is less than 3 height, (in case of AAA sequence the peak would be higher than 3, because of slightly higher fluorescent of A due to protocol peculiarities). The approximate ratio of G/A components in the 1072 position is equal.

Figure 7

Sanger sequencing and pyrosequencing of A/South Africa/3626/2013 virus of mice isolates (a): Sanger sequencing of PB2 gene fragment, (b): Detection of the nucleotide sequence in PB2 fragment 1064–1079 by pyrosequencing. Arrows show the peaks of interest.

Figure 7

Sanger sequencing and pyrosequencing of A/South Africa/3626/2013 virus of mice isolates (a): Sanger sequencing of PB2 gene fragment, (b): Detection of the nucleotide sequence in PB2 fragment 1064–1079 by pyrosequencing. Arrows show the peaks of interest.

Figure 8

Four circle diagram: correlations of four sets of virus properties—A (non-ts phenotype), B (toxicity for mice), C (thermal stability of the hemagglutinin), and D (pathogenicity for mice).