Compatibility of H9N2 avian influenza surface genes and 2009 pandemic H1N1 internal genes for transmission in the ferret model

Abstract

In 2009, a novel H1N1 influenza (pH1N1) virus caused the first influenza pandemic in 40 y. The virus was identified as a triple reassortant between avian, swine, and human influenza viruses, highlighting the importance of reassortment in the generation of viruses with pandemic potential. Previously, we showed that a reassortant virus composed of wild-type avian H9N2 surface genes in a seasonal human H3N2 backbone could gain efficient respiratory droplet transmission in the ferret model. Here we determine the ability of the H9N2 surface genes in the context of the internal genes of a pH1N1 virus to efficiently transmit via respiratory droplets in ferrets. We generated reassorted viruses carrying the HA gene alone or in combination with the NA gene of a prototypical H9N2 virus in the background of a pH1N1 virus. Four reassortant viruses were generated, with three of them showing efficient respiratory droplet transmission. Differences in replication efficiency were observed for these viruses; however, the results clearly indicate that H9N2 avian influenza viruses and pH1N1 viruses, both of which have occasionally infected pigs, have the potential to reassort and generate novel viruses with respiratory transmission potential in mammals.

Fig. 1.

Characteristics of H9N2:pH1N1 viruses in vitro. (A) Genes colored black come from pdmH1N1. Red genes originate from P10 (H9N2). Green genes are from WF10 (H9N2). (B) Replication of four reassortant viruses in MDCK cells. Six-well plates of confluent MDCK cells were inoculated with 0.1 multiplicity of infection of either 1P10 (red), 2P10 (green), 1WF10 (blue), or 2WF10 (black). Supernatants were harvested twice a day for 3 d and titered. Mean titer and SD were calculated. (C) Plaque morphology in MDCK cells for 1P10, 2P10, 1WF10, and 2WF10. Cells were infected with 1 × 10⁻⁶ dilution of stock virus except for the 1P10, in which a 1 × 10⁻⁵ dilution was used because of smaller plaque size.

Fig. 2.

Pathology produced by H9N2:pH1N1 viruses. Ferrets were inoculated with 1 × 10⁶ TCID₅₀ of either 1P10, 2P10, 1WF10, or 2WF10 and tissues were collected at 5 dpi. Samples were cut into 5-μm thick sections and stained using a standard H&E protocol by Histoserv Inc. (Magnification: 200×.)

Fig. 3.

Nasal wash titers from DI, DC, and RC ferrets. Ferrets (red lines) were infected with 1× 10⁶ TCID₅₀ of 1P10 (A), 2P10 (B), 1WF10 (C), or 2WF10 (D). At 1 dpi, one inoculated ferret was moved to a clean isolator with a naive ferret in direct contact (DC, blue lines). Additionally, another naive ferret was placed in the same isolator in a manner such that no direct contact was possible, only respiratory droplet contact (RC, green lines). Nasal washes were collected daily and titered in MDCK cells. Each virus was tested in duplicate.

Fig. 4.

Amino acid mutations during transmission of H9N2:pH1N1 viruses. Sequences from virus-positive nasal washes were generated for every ferret at the site in which a mutation was observed during peak shedding compared with the wild-type virus. Solid lines indicate days in which sequences were performed. Dotted lines indicated days in which viruses were detected but no sequences were generated (PB2 T58I DI and DC 2P10). Amino acids are indicated in color. A/V, T/I, and S/N indicate mixed virus population on the date shown.