Figure 1.
Infection of HAE by avian, but not human, influenza viruses is restricted at temperatures of the proximal airways.
(A) Comparison of multi-cycle virus growth in HAE inoculated with either A/Victoria/3/75 at 32°C (closed triangles) or 37°C (open triangles) and A/Dk/Eng/62 at 32°C (closed circles) or 37°C (open circles) both at MOI∼0.01. Apical viral titers at times shown were determined by standard plaque assay on MDCK cells. Data shown represents the mean titer +/−standard error (SE; n = 3–10 cultures). (B) Adenylate kinase activity released into the apical compartment of HAE over time after inoculation with A/Victoria/3/75 or A/Dk/Eng/62 at 32°C and 37°C as a measure of viral-induced CPE. Data shown represents the mean fold change over adenylate kinase activity derived from mock-inoculated HAE +/−SE (n = 3–8). Significance is noted (*p<0.05) where viral titers or AK levels obtained for A/Dk/Eng/62 at 32°C were statistically different from all other titers/AK measurements (Dk/37°C, Vic/32°C and Vic/37°C) at that particular time point. Significance is noted (†p<0.05) where AK levels obtained for A/Dk/Eng/62 at 32°C and 37°C were statistically different.
Figure 2.
Spread and histopathology of avian and human influenza viruses in HAE at temperatures of the proximal and distal airway.
(A) Representative en face photomicrographs of HAE inoculated with either A/Victoria/3/75 or A/Dk/Eng/62 at 32°C or 37°C, fixed at 6, 24, 48 and 72 hrs pi and stained for viral nucleoprotein (green) to determine numbers of cells infected. Scale bar equals 100 µm. (B) Representative histological cross-sections of HAE at 24, 72 and 120 hrs after inoculation with A/Victoria/3/75 or A/Dk/Eng/62 at 32°C or 37°C. H&E counterstain. Scale bar equals 20 µm.
Figure 3.
Temperature-dependent growth of different serotypes of influenza viruses in HAE.
Multi-step growth kinetics of (A) human influenza virus A/Eng/26/99 or (C) avian influenza virus A/Dk/Sing/97 (MOI∼0.1) at 32°C (open circles, dashed line) or 37°C (closed circles, solid line) in HAE +/−SE (n = 3 cultures). Multi-step growth kinetics in HAE inoculated with an MOI∼0.03 of (B) A/Udorn/307/72 (H3N2) or (D) A/VN/1203/04 (H5N1) at 33°C (open circles, dashed line) or 37°C (closed circles, solid line). Data represents mean titer across two different donors, each performed in duplicate +/−SE. Viral titers were determined by plaque assay in (A) and (B) and by TCID50 assay for (C) and (D). No significant differences in growth between temperatures were found for either A/Eng/26/99 or A/Udorn/307/72. A/VN/1203/04 was significantly restricted for growth at 24, 48 and 72 hrs pi (*p<0.05). (E) Representative histological cross-sections of HAE infected for 72 hrs at 37°C with A/Udorn/307/72 or A/VN/1203/04 and compared to mock-inoculated HAE. H&E counterstain. Scale bar equals 20 µm.
Figure 4.
Comparison of replication kinetics and spread of A/Victoria/3/75 with an avianized PB2, HA and/or NA to wild-type virus.
Multi-step growth kinetics in HAE inoculated with (Ai) PB2 polymerase mutant (K627E), (Bi) HA (L226Q, S228G) mutant, (Ci) Vic+Chick N1 reassortant virus, (Di) Vic (226–228)HA+Chick N1 at 32°C (solid line, closed circles) or 37°C (solid line, open circles). Wild-type A/Victoria/3/75 growth curves at 32°C (closed triangles) and 37°C (open triangles) are repeated in each panel and shown as dotted lines in (Ai), (Bi), (Ci) and (Di) for comparison. Data represent mean titer across 3–8 cultures +/−SE. Significance is noted (*p<0.05) where viral titer obtained for the mutant or reassortant virus at 32°C was statistically different from all other viral titers (mutant/37°C, wild-type/32°C and wild-type/37°C) at that time point. Significance is noted (†p<0.05) where viral titers obtained for the mutant / reassortant virus at 32°C and 37°C were statistically different. Quantification of numbers of cells infected (determined by en face staining for viral nucleoprotein) in HAE at 24 and 48 hrs pi at 32°C and 37°C for (Aii) PB mutant virus, (Bii) HA mutant virus and (Cii) N1 reassortant virus. Data obtained in parallel for wild-type A/Victoria/3/75 is repeated in each graph (striped bars) for comparison to the mutant (solid bars). Data shown represents the mean of the percentage of influenza virus antigen-positive epithelium across 10 different fields +/−SE. Differences in viral antigen positive epithelium between temperatures for each virus at 48 hrs pi is noted as significant (*p<0.05) or insignificant (NS). A one-way ANOVA model showed no significant differences between the wild-type virus and PB2 mutant at 32°C and 37°C at 48 hrs pi. (Dii) Representative en face photomicrographs of HAE inoculated with A/Victoria/3/75 or Vic (226–228)HA+Chick N1 at either 32°C or 37°C and stained for viral nucleoprotein (green) to determine numbers of cells infected 72 hrs pi. Scale bar represents 100 µm.
Figure 5.
Cell tropism of human, avian and avianized viruses in HAE.
Representative cross-sections of inoculated HAE, fixed 24 hrs pi, were probed for viral antigen (NP; green) and α−acetylated tubulin, a marker for ciliated cells (red). Notably, the staining pattern for wild-type A/Victoria/3/75 was identical to that of PR8+Vic HA/NA. Arrows mark ciliated cells infected with either wild-type A/Victoria/3/75 or PR8+Vic HA/NA; arrow-head denotes non-ciliated cells infected by these viruses. These data indicate that viruses with Victoria glycoproteins were able to infect both cell types previously shown to express α2,6 SA [13]. Viral antigen was detected only in ciliated cells in cultures inoculated with Vic-226-228HA (in the Victoria background with either endogenous N2 or avian N1 or PR8+Chick HA/NA). Scale bar equals 20 µm.
Figure 6.
Temperature restriction of avian influenza viruses at 32°C can be mimicked by inserting avian envelope glycoproteins into human influenza viruses.
(A) Multi-step growth kinetics initiated in HAE over time with PR8+Vic HA/NA at 32°C (closed triangles) or 37°C (open triangles) and PR8+Chick HA/NA at 32°C (closed circles) or 37°C (open circles) in HAE. Apical viral titers were determined at the times shown by standard plaque assay. Data shown represents mean titer across 4–8 cultures +/−SE. (B) Adenylate kinase activity in apical washes of virus-infected HAE expressed as fold-change over adenylate kinase activity in mock-inoculated HAE +/−SE (n = 4–8). Significance is noted (*p<0.05) where viral titers or AK levels obtained for PR8+Chick HA/NA at 32°C were statistically different from all other titers/AK measurements (Chick/37°C, Vic/32°C and Vic/37°C) at that particular time point. Significance is noted (†p<0.05) where AK levels obtained for PR8+Chick HA/NA at 32°C and 37°C were statistically different. (C,D) Representative en face photomicrographs of viral nucleoprotein immunoreactivity (green) in HAE inoculated with (C) PR8+Vic HA/NA or (D) PR8+Chick HA/NA, at 24, 48 and 72 hrs pi at 32°C (lower rows) or 37°C (upper rows).