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.

A Global Perspective on H9N2 Avian Influenza Virus

“…Un-adapted, H9N2 viruses do not transmit efficiently between pigs, and swine H9N2 isolates show little evidence of mammalian adaption suggesting repeated reintroduction from avian hosts rather than continuous within-species circulation [159,160]. Repeated serial passage through pigs can lead to partial adaptation allowing for modest replication and transmission [160].…”

“…Un-adapted, H9N2 viruses do not transmit efficiently between pigs, and swine H9N2 isolates show little evidence of mammalian adaption suggesting repeated reintroduction from avian hosts rather than continuous within-species circulation [159,160]. Repeated serial passage through pigs can lead to partial adaptation allowing for modest replication and transmission [160]. Although H9N2 viruses don't appear to actively circulate in pigs, there remains a possibility that these viruses could spill over into these hosts due to the proximity between poultry and pigs in many smallholding farms leading to the potential for reassortment with currently circulating swine influenza viruses.…”

H9 Influenza Viruses: An Emerging Challenge

“…H9 IAVs do not seem to affect pigs, causing just mild respiratory signs. Most H9 isolates from pigs have been from farms in Hong Kong and China (Xu et al 2004;Cong et al 2007;Yu et al 2011a;Wang et al 2016b) and appear to be related to recurrent reintroductions rather than intraspecies circulation (Mancera Gracia et al 2017b;Yang et al 2017b). These observations are in line with experiments in pigs that show impaired transmission of H9N2 strains in pigs unless provided as reassortants in the background of swine-origin or human-origin IAV strains (Obadan et al 2015;Wang et al 2016b;Mancera Gracia et al 2017a,b;Yang et al 2017b).…”

Genetic determinants of receptor-binding preference and zoonotic potential of H9N2 avian influenza viruses

“…Several of the residues identified in this study have been previously described as directly or indirectly affecting H9 receptor binding, including position 155, 190, 225, 226 and 227 [13, 17–20, 30, 31]. In previous studies, generally only one or two of these residues are measured in isolation for their receptor binding effect or are tested in a non- or semi-quantitative manner.…”