The production and development of H7 Influenza virus pseudotypes for the study of humoral responses against avian viruses

Abstract

In recent years, high pathogenicity avian influenza (HPAI) virus, H5N1, low pathogenicity avian influenza (LPAI) virus, H9N2, and both HPAI and LPAI H7 viruses have proved devastating for the affected economies reliant on poultry industry, and have posed serious public health concerns. These viruses have repeatedly caused zoonotic disease in humans, raising concerns of a potential influenza pandemic. Despite the focus on the HPAI H5N1 outbreak in 1997 some H7 strains have also shown to be occasionally adaptable to infecting humans. Therefore, applying knowledge of the H5 virus evolution and spread to the development of sensitive serological methods is likely to improve our ability to understand and respond to the emergence of other HPAI and LPAI viruses, present within the avian populations, with the potential to infect humans and other species. In the present study we describe the construction and production of lentiviral pseudotypes bearing envelope glycoproteins of LPAI and HPAI H7 avian influenza viruses, which have been responsible for several outbreaks in the past decade. The H7 pseudotypes were evaluated in pseudotype-based neutralization (pp-NT) assays in order to detect and quantify the presence of neutralizing antibodies in avian sera, which were confirmed H7 positive by inhibition of haemagglutination (HI) test. Overall, our results substantiate influenza virus pseudotype neutralization as a robust tool for influenza sero-surveillance.

Keywords: H7 lentiviral pseudotype; HPAI; LPAI; micro-neutralization; pandemic potential.

Figure 1.

Infectivity of pseudotyped lentiviral vectors carrying HA glycoprotein of HPAI A/chicken/Italy/13474/1999, A/chicken/Pakistan/34668/1995, H7 A/chicken/FPV/Rostock/34 and LPAI H7N1 A/chicken/1082/1999. Influenza pseudotypes were produced by adding exogenous neuraminidase 24hr after transfection (for H7 A/chicken/Italy/13474/1999, additionally an NA plasmid has been used). The transduction titers of the released pseudotypes were expressed as the mean ±SD of relative luciferase units (RLUs).

Figure 2.

Panel of H7 A/chicken/Italy/13474/1999 HA pseudotypes produced in combination with different NA plasmids (N1 from A/chicken/Italy/13474/1999, N3 from A/chicken/Pakistan/34668/1995 and N7 from A/chicken/Netherlands/1/2003). Comparison of transduction efficiency in target HEK 293T cells. Titers of released pseudotypes were expressed as the mean ±SD of relative luciferase units (RLUs).

Table 1.

Comparison of neutralization activity of chicken immune sera (positive for the H7N1 vaccine strain: A/chicken/Italy/1067/1999) against three different H7 pseudotypes: A/ck/Italy/1082/1999, A/ck/Italy/13474/1999 and A/chicken/FPV/Rostock/34 (Set A). H7 Influenza pseudotypes bearing HA from A/ck/Italy/13474/1999 were also used for testing neutralizing activity of a panel of sera (Set B: No. 8–No. 17) collected from naturally infected turkeys (positive for H7N3 A/turkey/Italy/9289/V02). Additional sera previously found H5 positive by HI have been tested (Set C). A non-linear regression approach, dose-response inhibition has been used for the analysis). Values are reported as 90% inhibitory concentration (IC₉₀). Haemagglutination inhibition titres are also reported.