Effect of serial pig passages on the adaptation of an avian H9N2 influenza virus to swine

Abstract

H9N2 avian influenza viruses are endemic in poultry in Asia and the Middle East. These viruses sporadically cause dead-end infections in pigs and humans raising concerns about their potential to adapt to mammals or reassort with human or swine influenza viruses. We performed ten serial passages with an avian H9N2 virus (A/quail/Hong Kong/G1/1997) in influenza naïve pigs to assess the potential of this virus to adapt to swine. Virus replication in the entire respiratory tract and nasal virus excretion were examined after each passage and we deep sequenced viral genomic RNA of the parental and passage four H9N2 virus isolated from the nasal mucosa and lung. The parental H9N2 virus caused a productive infection in pigs with a predominant tropism for the nasal mucosa, whereas only 50% lung samples were virus-positive. In contrast, inoculation of pigs with passage four virus resulted in viral replication in the entire respiratory tract. Subsequent passages were associated with reduced virus replication in the lungs and infectious virus was no longer detectable in the upper and lower respiratory tract of inoculated pigs at passage ten. The broader tissue tropism after four passages was associated with an amino acid residue substitution at position 225, within the receptor-binding site of the hemagglutinin. We also compared the parental H9N2, passage four H9N2 and the 2009 pandemic H1N1 (pH1N1) virus in a direct contact transmission experiment. Whereas only one out of six contact pigs showed nasal virus excretion of the wild-type H9N2 for more than four days, all six contact animals shed the passage four H9N2 virus. Nevertheless, the amount of excreted virus was significantly lower when compared to that of the pH1N1, which readily transmitted and replicated in all six contact animals. Our data demonstrate that serial passaging of H9N2 virus in pigs enhances its replication and transmissibility. However, full adaptation of an avian H9N2 virus to pigs likely requires an extensive set of mutations.

Fig 1. Nasal virus excretion of A/quail/Hong Kong/G1/1997 influenza virus during ten blind serial pig passages.

Two pigs (solid line and dashed line) were involved in each passage, each line represents the individual virus titers in nose swabs. The detection limit of the test is indicated with a dotted line at 1.7 log₁₀ TCID₅₀/100 mg of secrete.

Fig 2. Nasal virus excretion and direct contact transmission of A/Qa/HK/P0, A/Qa/HK/P4 and A/Cal/04/09 influenza viruses in pigs.

Three pigs were intranassally inoculated with 6.5 log₁₀ TCID₅₀ of the indicated virus per pig and individually housed in different isolators. Forty-eight hours later, two direct contact animals were co-housed with the inoculated pigs. Each graphic is identified with a two digits number: the first one corresponds to the isolator number and the second to the virus tested. Therefore, each column represents a different virus and each row represents a different isolator. The detection limit (dotted line) of the test was 1.7 log₁₀ TCID₅₀/100 mg of secrete.

Fig 3. The effect of the serial passaging on the amount of virus produced over the course of the experiment using a single-step growth assay in MDCK cells.

Each data point on the curve is the mean ± SD of the three independent experiments.

Fig 4. Alignment of the deduced amino acid sequences in the HA of all swine H9N2 isolates (n = 47) available in Genbank on 22^nd August 2016.

The name of each strain included in the analysis is followed by the accession number between brackets. Residues at positions 190, 225, 226 and 228 are highlighted in gray. Amino acids that are different from those in A/quail/Hong Kong/G1/1997 are shown, conserved residues are shown as dots.

Fig 5. Alignment of deduced amino acid sequences in the HA of all human H9N2 isolates (n = 12) available in Genbank on 22^nd August 2016.

Strain names are followed by accession numbers (between brackets). Residues at positions 190, 225, 226 and 228 are highlighted in gray. Amino acids that are different from those in A/quail/Hong Kong/G1/1997 are shown, conserved residues are shown as dots.