Use of fractional factorial design to study the compatibility of viral ribonucleoprotein gene segments of human H7N9 virus and circulating human influenza subtypes

Abstract

Avian H7N9 influenza viruses may pose a further threat to humans by reassortment with human viruses, which could lead to generation of novel reassortants with enhanced polymerase activity. We previously established a novel statistical approach to study the polymerase activity of reassorted vRNPs (Influenza Other Respir Viruses. 2013;7:969-78). Here, we report the use of this method to study recombinant vRNPs with subunits derived from human H1N1, H3N2, and H7N9 viruses. Our results demonstrate that some reassortant vRNPs with subunits derived from the H7N9 and other human viruses can have much higher polymerase activities than the wild-type levels.

Keywords: Fractional factorial design; H7N9; influenza polymerase.

Figure 1

Mean relative polymerase activity of chimeric vRNPs of different viruses from the 27-run fractional factorial design at different temperatures. Twenty-seven chimeric vRNP combinations originated from human H1N1 (H1), human H3N2 (H3), and human isolate of avian H7N9 (H7) viruses were selected from the 27-run fractional factorial design. The chimeric vRNPs were reconstituted in 293T cells at 33°C (black) and 37°C (white). Mock transfection (M) and wild-type H1N1, H3N2, and H7N9 vRNPs were used as control. The polymerase activities of the chimeric vRNPs were studied by luciferase reporter assay. The normalized data are expressed as mean relative polymerase activity in relative to the polymerase activity of wild-type H3N2 vRNP at the corresponding temperatures (n = 3). Error bars represent one standard deviation.

Figure 2

Mean relative polymerase activity of wild-type and chimeric vRNPs at different temperatures. Wild-type (A) H1N1 (H1), (B) H3N2 (H3), or (C) H7N9 (H7) and chimeric vRNPs with different subunits were reconstituted in 293T cells at 33°C (black) and 37°C (white). Mock-treated cells (M) were used as controls. The polymerase activities of the chimeric vRNPs were determined by luciferase reporter assay. The normalized data are expressed as mean relative polymerase activity in relative to the polymerase activity of wild-type (A) H1N1, (B) H3N2 or (C) H7N9 vRNP at the corresponding temperatures, respectively (n = 3). Error bars represent one standard deviation. (*P < 0·01, **P < 0·05, by t-test).

Figure 3

Mean relative polymerase activity of wild-type and chimeric vRNPs at different temperatures. Wild-type (A) H1N1 (A/HK/4259592/2013; H1′), (B) H3N2 (A/HK/4003076/2013; H3′), or (C) H7N9 (H7) and chimeric vRNPs with different subunits were reconstituted in 293T cells at 33°C (black) and 37°C (white). Mock-treated cells (M) were used as controls. The polymerase activities of the chimeric vRNPs were determined by luciferase reporter assay. The normalized data are expressed as mean relative polymerase activity in relative to the polymerase activity of wild-type (A) H1N1, (B) H3N2, or (C) H7N9 vRNP at the corresponding temperatures, respectively (n = 3). Error bars represent one standard deviation (*P < 0·01 by t-test).