1. Microbiology and Infectious Disease
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HA stabilization promotes replication and transmission of swine H1N1 gamma influenza viruses in ferrets

  1. Meng Hu
  2. Guohua Yang
  3. Jennifer DeBeauchamp
  4. Jeri Carol Crumpton
  5. Hyunsuh Kim
  6. Lei Li
  7. Xiu-Feng Wan
  8. Lisa Kercher
  9. Andrew S Bowman
  10. Robert G Webster
  11. Richard J Webby
  12. Charles J Russell  Is a corresponding author
  1. Department of Infectious Diseases, St. Jude Children's Research Hospital, United States
  2. Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, United States
  3. Missouri University Center for Research on Influenza Systems Biology (CRISB), University of Missouri, United States
  4. Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, United States
  5. Bond Life Sciences Center, University of Missouri, United States
  6. Department of Electrical Engineering Computer Science, College of Engineering, University of Missouri, United States
  7. MU Informatics Institute, University of Missouri, United States
  8. Department of Veterinary Preventive Medicine, The Ohio State University, United States
  9. Department of Microbiology, Immunology & Biochemistry, College of Medicine, The University of Tennessee Health Science Center, United States
Research Article
Cite this article as: eLife 2020;9:e56236 doi: 10.7554/eLife.56236
8 figures, 2 tables and 2 additional files

Figures

Figure 1 with 2 supplements
HA activation pH values and replication capacities of H1N1 pandemic (pdm) and gamma virus isolates from humans and swine.

(A) HA activation pH values. Vero cells were infected with IAVs (human pdm n = 21; swine pdm n = 12; swine gamma n = 22) at a multiplicity of infection (MOI) of 3 PFU/cell. At 16 hr post-infection, HA activation pH values were measured by syncytia assay. Each symbol represents the mean HA activation pH value of an individual isolate. A dashed line is shown at pH 5.5. (B) Replication capacities in MDCK cells. MDCK cells were infected by pandemic and gamma viruses at an MOI of 0.01 PFU/cell. Cell culture supernatants were harvested at 12, 24, and 36 hr, and were titrated by TCID50 on MDCK cells. Each symbol represents the mean peak titer of an individual isolate. The white square in the human pdm group is the reference virus A/TN/09. All experiments were independently performed at least twice. P values were determined according to one-way ANOVA followed by a Tukey’s multiple comparisons test.

Figure 1—figure supplement 1
Phylogenic analyses of HA segments of swine and human H1N1 influenza A viruses (2009–2016).

HA amino acids of viruses used in this study were obtained by in-house Sanger sequencing or retrieved from GenBank. Full-length HA segments of representative human and swine isolates in 1918–2016 were retrieved from GenBank. Phylogeny of HA segments was constructed by using Tree software displaying human isolates in this study in blue, swine isolates in this study in red, and other representative isolates from GenBank in gray.

Figure 1—figure supplement 2
Virus replication in MDCK cells.

The virus-infection experiment was performed as described in Figure 1. Each symbol represents the mean virus titer of an individual isolate detected at the reported time. The experiments were independently performed at least twice. Statistical significance was determined by one-way ANOVA followed by a Tukey’s multiple comparisons.

Genetic, structural, and sequence analyses of swine gamma Pair 1 and Pair 2 viruses.

(A) Lineages of Pair 1 and 2 viruses. Virus gene segments were analyzed by using CLC Genomics Workbench version 11.0.1. A/Ohio/09/2015 and A/Iowa/39/2015 H1N1 viruses are gamma viruses from human infections (Pulit-Penaloza et al., 2018). Gene segments from pandemic and swine endemic are marked in black and white, respectively. (B) HA protein structure and locations of HA residue variations. HA1 residue 210 (yellow) is located in the HA receptor-binding domain head (green) in contact with an adjacent protomer (gray). HA2 residue 117 (yellow) is located in the stalk region (red) in contact with the fusion peptide (black) of an adjacent protomer. HA1 residues in the stalk are colored blue, and two protomers of the trimer are colored gray. The structure was generated by Mac PyMOL using A/California/04/2009 (H1N1) (PDB entry 3UBE). (C) Sequence variations between Pair 1 isolates G15 and P4. (D) Sequence variations between Pair 2 isolates P19 and P24. Whole-genome analyses were performed by next-generation sequencing. Amino-acid variations with frequencies greater than 5% are shown in colored pie charts. H3 numbering was used for the HA segment.

Pair 1 (G15 and P4) virus characterization in vitro.

(A) HA activation pH measured by syncytia assay. Viruses were inoculated into Vero cells at an MOI of 3 PFU/cell. Recombinant viruses with HA and NA segments from G15 and P4 were rescued in the background of the six internal genes from A/TN/09 using reverse genetics. Representative images of three independent experiment results are shown. (B) HA inactivation pH measured by loss of infectivity as a function of acid exposure. Virus aliquots were treated with pH-adjusted PBS, re-neutralized, and subjected to measurement of residual virus infectivity by TCID50. (C) Virus growth capacity in swine testicular (ST) and MDCK cells. Viruses were inoculated into ST and MDCK cells at an MOI of 0.01 PFU/cell. Cell culture supernatants were harvested at the indicated time points and quantified by TCID50. (D) Binding specificities toward α(2, 6)- or α(2, 3)-linked sialic acid receptors. Viruses were inoculated onto fetuin-coated plates. Virus binding affinities toward α2,6- or α2,3-linked sialylglycopolymers were measured by solid-phase receptor binding assay. Recombinant A/Puerto Rico/9/1934 (H1N1) with the HA segment from A/Mallard/Alberta/383/2009 (H5N1) was used as a control. All data were means ± SD of at least two independent experiments.

Pair 2 (P19 and P24) virus characterization in vitro.

(A) Syncytia assay results for wild-type and 6+2 reassortant viruses. (B) HA inactivation pH values quantified by TCID50. (C) Virus replication in ST and MDCK cells inoculated at an MOI of 0.01 PFU/cell and quantified by TCID50. (D) Receptor binding specificities to α2,6- or α2, 3-linked sialic acid receptors. All data were mean ± SD of at least two independent experiments.

Figure 5 with 1 supplement
Pair 1 (G15 and P4) replication and transmission in ferrets.

Three inoculated (Donor) ferrets were caged separately. On the next day, 3 naive Contact ferrets each were cohoused with the Donor and 3 naive Airborne ferrets were placed into perforated cages allowing transmission through the air. (A–C) Virus TCID50 titers of nasal washes from Donor (A), Contact (B), and (C) Airborne ferrets. Each bar represents the virus titer of an individual sample, and the dashed lines represent the limit of detection. (D) HAI titers of day-21 sera of ferrets infected or exposed to the tested viruses. The difference of 3/3 versus 1/3 airborne-transmission events was not statistically significant (p>0.05).

Figure 5—figure supplement 1
Clinical symptoms in ferrets upon infection with Pair 1 viruses.

Three ferrets each were intranasally inoculated with 106 PFU of tested viruses and were caged separately. Ferret body weight and temperature were measured daily until day 14. (A) Comparison of body temperature of ferrets infected with Pair 1 viruses (G15 and P4). (B) Comparison of body weight changes of ferrets infected with Pair 1 viruses. *p<0.05, according to two-tailed Student’s t-test.

Figure 6 with 1 supplement
Phenotypes and genotypes of Pair 1 (G15 and P4) viruses after infection and transmission in ferrets.

The transmission study was performed as described in Figure 5. (A) Virus HA activation pH values (means ± SD) measured by at least two independent syncytia assay experiments. (B) HA variant percentages in G15 and P4 viruses after infection and contact transmission in ferrets calculated by using mutations (synonymous and nonsynonymous) with a frequency larger than 5% during the transmission study in comparison to the consensus sequences of the initial inoculums. P values were determined by Mann-Whitney U test. (C) Proportions of HA1-N210 and HA1-S210 in G15 before and after infection and transmission in ferrets.

Figure 6—figure supplement 1
Heat maps of mutations detected in nasal washes of Pair 1 ferrets.

Virus whole-genome sequences of nasal washes were obtained for P4 Donor ferrets (A), P4 Contact ferrets (B), P4 Airborne ferrets (C), G15 Donor ferrets (D), G15 Contact ferrets (E) and G15 Airborne ferrets (F). Samples were obtained and analyzed as described in Figure 6. Mutations with frequencies greater than 5% are shown.

Figure 7 with 1 supplement
Pair 2 (P19 and P24) replication and transmission in ferrets.

The transmission study was performed as described in Figure 5. (A–C) Virus TCID50 titers of nasal washes from Donor (A), Contact (B), and (C) Airborne ferrets. Each bar represents the virus titer of an individual sample, and the dashed lines represent the limit of detection. (D) HAI titers of day-21 sera of ferrets infected or exposed to the tested viruses. The difference of 1/3 versus 0/3 airborne-transmission events was not statistically significant (p>0.05).

Figure 7—figure supplement 1
Clinical symptoms in ferrets upon infection with Pair 2 viruses.

Three ferrets each were intranasally inoculated with 106 PFU of tested viruses and were caged separately. Ferret body weight and temperature were measured daily until day 14. (A) Comparison of body temperature of ferrets infected with Pair 2 viruses (P19 and P24). (B) Comparison of body weight changes of ferrets infected with Pair 2 viruses. *p<0.05, according to two-tailed Student’s t-test.

Figure 8 with 1 supplement
Phenotypes and genotypes of Pair 2 (P19 and P24) viruses after infection and transmission in ferrets.

Virus phenotypes and genotypes were determined as described in Figure 6. (A) Virus HA activation pH values (means ± SD) measured by at least two independent syncytia assay experiments. P values were determined by one-way ANOVA followed by a Tukey’s multiple comparisons test. (B) HA variant percentages in P19 and P24 viruses after infection and contact transmission in ferrets. P values were determined by Mann-Whitney U test. (C) Proportions of HA2-T117 and HA2-N117 in P19 before and after infection and transmission in ferrets.

Figure 8—figure supplement 1
Heat maps of mutants detected in nasal washes of Pair 2 infected and/or exposed ferrets during and after transmission.

Virus whole-genome sequences of nasal washes collected in P24 Donor ferrets (A), P24 Contact ferrets (B), P19 Donor ferrets (C), P19 Contact ferrets (D) and P19 Airborne ferrets (E). Samples were obtained and analyzed as illustrated in Figure 6. Mutations with frequencies greater than 5% are shown.

Tables

Table 1
H1N1 swine gamma virus characterization before and after infection and transmission in ferrets.
Virus characterizationPair 1 virusesPair 2 viruses
G15P4P19P24
HA activation pH*Wild-type viruses5.85.55.95.6
Recombinant (6+2)5.85.56.05.6
HA inactivation pHWild-type viruses5.85.65.95.7
Genome variationsHA1-N210
PB2-L648
PA-P271
HA1-S210
PB2-M648
PA-T271
HA2-T117HA2-N117
Replication in vitroST cellsSimilarSimilar
MDCK cellsSimilarSimilar
Receptor binding specificity§α2, 6LowerHigherSimilar
α2, 3HigherLowerSimilar
Donor ferretsViruses isolated3/33/33/33/3
Seroconversion3/33/33/33/3
Day of peak titer32.3 (±1.2)4.3 (±1.2)1.7 (±1.2)
P (t-test), peak titer day0.210.047
Peak titers (log10 TCID50)6.4 (±0.54)6.7 (±0.45)6.6 (±0.19)6.9 (±0.89)
HA activation pH range5.6–5.95.5–5.65.6–6.05.5-.6
Major mutantsHA1-N210SNoneHA2-T117NNone
Contact ferretsViruses isolated3/33/33/33/3
Seroconversion3/33/33/33/3
HA activation pH range5.6–5.95.5–5.65.6–5.95.6–5.7
Major mutantsHA1-N210SNoneHA2-T117NNone
Airborne ferretsViruses detected2/31/31/30/3
Seroconversion3/31/31/30/3
HA activation pH5.65.55.6NA**
Major variantsHA1-N210SNoneHA2-T117NNA
  1. *HA activation pH measured by syncytia assay.

    Recombinant 6+2 viruses contained HA and NA genes from swine gamma isolates and the six internal genes from A/TN/1-560/09 (H1N1).

  2. HA inactivation pH measured by acid-induced inactivation pH with TCID50 readout.

    §Receptor binding specificity measured by solid-phase receptor binding assay.

  3. HA activation pH of airborne-transmitted virus on first day of isolation.

    **NA, Not applicable.

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Animal (Mustela putorius furo)Fitch ferretTriple F Farmsmale, 5–6 months old
Cell line (Canis lupus familiaris)MDCK, Madin-Darby Canine Kidney epithelialATCCATCC Cat# CCL-34, RRID:CVCL_0422
Cell line (Chlorocebus sabaeus)Vero, African green monkey kidney epithelialATCCATCC Cat# CCL-81, RRID:CVCL_0059
Cell line (Sus scrofa)Swine testis (ST) fibroblastATCCATCC Cat# CRL-1746, RRID:CVCL_2204
Cell line (Homo sapiens)293T/17 [HEK 293T/17], Human epithelial kidney expressing SV40 large T antigenATCCATCC Cat# CRL-11268, RRID:CVCL_1926
Recombinant DNA reagentpHW2000 A/TN/2009 cDNA reverse genetics plasmidsRussier et al., 2016PMID:26811446
Recombinant DNA reagentpHW2000 swine gamma HA and NA cDNA reverse genetics plasmidsThis paper
SoftwareCLC Genomics Workbench 11.0.1Qiagen
SoftwarePrism 7GraphPad
Strain, strain background (Influenza A virus)A/Tennessee/1-560/2009 (H1N1)NCBINCBI:txid646491
Strain, strain background (Influenza A virus)A/swine/Illinois/2A-1213-G15/2013 (H1N1) HAGenbankMT533249A_sw_IL_1213G15_13_HA
Strain, strain background (Influenza A virus)A/swine/Illinois/2B-0314-P4/2014 (H1N1) HAGenbankMT533251A_sw_IL_2B0314P4_14_HA
Strain, strain background (Influenza A virus)A/swine/Illinois/2E-0113-P19/2013 (H1N1) HAGenbankMT533255A_sw_IL_2E0113P19_13_HA
Strain, strain background (Influenza A virus)A/swine/Illinois/2E-0113-P24/2013 (H1N1) HAGenbankMT533252A_sw_IL_2E0113P24_13_HA

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