Human HA and polymerase subunit PB2 proteins confer transmission of an avian influenza virus through the air

Abstract

The influenza virus genes that confer efficient transmission of epidemic and pandemic strains in humans have not been identified. The rapid spread and severe disease caused by the 1918 influenza pandemic virus makes it an ideal virus to study the transmissibility of potentially pandemic influenza strains. Here, we used a series of human 1918-avian H1N1 influenza reassortant viruses to identify the genetic determinants that govern airborne transmission of avian influenza viruses. We have demonstrated that the 1918 HA gene was necessary for efficient direct contact transmission, but did not allow respiratory droplet transmission between ferrets of an avian influenza virus possessing an avian polymerase subunit PB2. The 1918 PB2 protein was found to be both necessary and sufficient for airborne transmission of a virus expressing the 1918 HA and neuraminidase. Also, it was found that influenza viruses that were able to transmit efficiently in ferrets were able to replicate efficiently at the lower temperature (33 degrees C) found in the environment of mammalian airway. These findings demonstrate that the adaptation of the HA and PB2 proteins are critical for the development of pandemic influenza strains from avian influenza viruses.

Fig. 1.

Direct contact transmissibility of wild-type, mutant, and reassortant avian H1N1 viruses. We inoculated 3 ferrets with 10⁶ EID₅₀ of Dk/NY/96 (A), Dk/NY/96-DD (B), 1918NA:Dk/NY/96-DD (C), or 1918HANA:Dk/NY/96 (D). One day p.i., a naive ferret was placed in each cage to establish contact between the animal pairs. Viral replication was monitored by titration of NWs collected every other day in both inoculated (Left) and contact (Right) animals. The limit of detection was 10^1.5 EID₅₀/mL, and is indicated by a solid line on each representation.

Fig. 2.

Respiratory droplet transmissibility of 1918-Dk/NY/96 reassortant H1N1 viruses. We inoculated 3 ferrets with 10⁶ EID₅₀ of 1918-DK/NY/96 reassortant viruses containing the 1918 HA and NA (A), the 1918 HA, NA, PB1, PB2, and PA (B), the 1918 HA, NA, and PB1 (C), the 1918 HA, NA, and PA (D), and the1918 HA, NA, and PB2 (E). Also, the transmissibility of a 1918 virus containing the Dk/NY/96 PB2 gene was examined (F). All animals were housed individually in specialized cages that permit exchange of respiratory droplets, but prevent direct or indirect contact between inoculated-contact animal pairs. One day p.i., a naive contact animal was placed in each adjacent cage. Viral replication was monitored by titration of NWs collected every other day in both inoculated (Left) and contact (Right) animals. The limit of detection was 10^1.5 EID₅₀/mL, and is indicated by a solid line on each representation.

Fig. 3.

Respiratory droplet and contact transmissibility of 1918-PB2-K627E. The 1918-PB2K627E mutant virus was tested in respiratory droplet (A) and direct contact (B) transmission experiments. In both cases, ferrets were inoculated with 10⁶ EID₅₀ of 1918-PB2-K627E. Viral replication was monitored by titration of NWs collected every other day in both inoculated (Left) and contact (Right) animals. The limit of detection was 10^1.5 EID₅₀/mL, and is indicated by a solid line on each representation.

Fig. 4.

Plaque morphology of MDCK cells infected with H1N1 reassortant viruses. Confluent monolayers of MDCK cells were inoculated with the indicated H1N1 reassortant viruses, incubated at either 37 °C or 33 °C for 48 h, and visualized for plaque morphology.