Virologic Differences Do Not Fully Explain the Diversification of Swine Influenza Viruses in the United States

Abstract

Influenza A(H1N1) viruses entered the U.S. swine population following the 1918 pandemic and remained genetically stable for roughly 80 years. In 1998, there was an outbreak of influenza-like illness among swine that was caused by A(H3N2) viruses containing the triple reassortant internal gene (TRIG) cassette. Following the TRIG cassette emergence, numerous reassortant viruses were isolated in nature, suggesting that the TRIG virus had an enhanced ability to reassort compared to the classical swine virus. The present study was designed to quantify the relative reassortment capacities of classical and TRIG swine viruses. Reverse genetic viruses were generated from the classical H1N1 virus A/swine/MN/37866/1999 (MN/99), the TRIG virus A/swine/NC/18161/2002 (NC/02), and a seasonal human H3N2 virus, A/TX/6/1996 (TX/96), to measure in vitro reassortment and growth potentials. After coinfection with NC/02 or MN/99 plus TX/96, H1/H3 double-positive cells were identified. Delayed TX/96 infection was fully excluded by both swine viruses. We then analyzed reassortant H3 viruses. Seventy-seven of 81 (95.1%) TX/96-NC/02 reassortants contained at least one polymerase gene segment from NC/02, whereas only 34 of 61 (55.7%) MN/99-TX/96 reassortants contained at least one polymerase gene segment from MN/99. Additionally, 38 of 81 (46.9%) NC/02-TX/96 reassortants contained all NC/02 polymerase gene segments, while none of the MN/99-TX/96 reassortants contained all MN/99 polymerase genes. There were 21 H3 reassortants between MN/99 and TX/96, compared to only 17 H3 reassortants between NC/02 and TX/96. Overall, the results indicate that there are no distinct differences in the ability of the TRIG to reassort with a human virus compared to the classical swine virus.

Importance: There appear to be no differences in the abilities of classical swine and TRIG swine viruses to exclude a second virus, suggesting that under the right circumstances both viruses have similar opportunities to reassort. The increased percentage of TRIG polymerase gene segments in reassortant H3 viruses indicates that these viruses may be more compatible with gene segments from other viruses; however, this needs to be investigated further. Nevertheless, the classical swine virus also showed the ability to reassort, suggesting that factors other than reassortment capacity alone are responsible for the different epidemiologies of TRIG and classical swine viruses. The post-TRIG diversity was likely driven by increased intensive farming practices rather than virologic properties. Our results indicate that host ecology can be a significant factor in viral evolution.

FIG 1

Flow cytometry dot plots of MDCK cells coinfected at an MOI of 0.01. Following infections, the cells were stained with fluorescently labeled anti-H1 and anti-H3 monoclonal antibodies and quantified by flow cytometry. The cells were gated based on viable MDCK cells, and limits were set based on single-stained cell populations. Percentages were calculated based on the numbers of labeled cells divided by the total number of gated cells. (A and B) There was no viral exclusion in MDCK cells simultaneously infected with MN/99 and TX/96 (A) or with NC/02 and TX/96 (B). (C and D) H3 virus (TX/96) was fully excluded when infection was delayed until 24 h after H1 infection with MN/99 (C) or NC/02 (D). (E and F) H1 virus was fully excluded when infection was delayed until 24 h after H3 (TX/96) infection for both MN/99 (E) and NC/02 (F).

FIG 2

Viral replication kinetics in pSRECs infected at an MOI of 0.01. Following infection, samples were collected at 10, 12, 14, 16, 18, 20, 24, 36, and 48 hpi and stored at −80°C until titration. The data represent 2 independent experiments with viruses titrated in triplicate. There was no significant difference in the growth of the reverse genetic parental viruses and the 6 + 2 reassortant viruses with the H3 HA gene and the N2 NA gene. The mock-infected cells were treated with infection medium only.

FIG 3

Viral titers in inoculated and direct contact pigs. The horizontal dashed lines indicate the limit of detection. All nasal swabs were collected at 1, 3, 5, 7, 9, and 11 dpi and stored at −80°C until titration. The titers were determined in triplicate and calculated based on the Reed-Muench method (25). (A) Growth and transmission of the human TX/96 virus in pigs. No contact transmission occurred. (B) Growth and transmission of the classical swine virus with the human HA and NA genes and the TRIG PB1 gene. Again, no transmission occurred. (C) Growth and transmission of the classical swine virus with the human HA and NA genes. All 3 contact animals became infected by 5 dpi. (D) Growth and transmission of the TRIG swine virus with the human HA and NA genes. Again, all 3 contact animals became infected by 5 dpi.