Elsevier

Vaccine

Volume 28, Issue 36, 16 August 2010, Pages 5803-5809
Vaccine

Repository of Eurasian influenza A virus hemagglutinin and neuraminidase reverse genetics vectors and recombinant viruses

https://doi.org/10.1016/j.vaccine.2010.06.072Get rights and content

Abstract

Reverse genetics can be used to produce recombinant influenza A viruses containing virtually every desired combination of hemagglutinin (HA) and neuraminidase (NA) genes using the virus backbone of choice. Here, a repository of plasmids and recombinant viruses representing all contemporary Eurasian HA and NA subtypes, H1–H16 and N1–N9, was established. HA and NA genes were selected based on sequence analyses of influenza virus genes available from public databases. Prototype Eurasian HA and NA genes were cloned in bidirectional reverse genetics plasmids. Recombinant viruses based on the virus backbone of A/PR/8/34, and containing a variety of HA and NA genes were produced in 293T cells. Virus stocks were produced in MDCK cells and embryonated chicken eggs. These plasmids and viruses may be useful for numerous purposes, including influenza virus research projects, vaccination studies, and to serve as reference reagents in diagnostic settings.

Introduction

Avian influenza A viruses are classified on the basis of antigenic variation of two glycoproteins expressed on the surface of virus particles, hemagglutinin (HA) and neuraminidase (NA). In wild birds throughout the world, influenza A viruses representing 16 HA and 9 NA subtypes have been detected, which can be found in numerous combinations (e.g., H1N1, H16N3) [1], [2]. Aquatic birds, in particular those belonging to the orders Anseriformes (mostly ducks, geese and swans) and Charadriiformes (mostly gulls, terns and shorebirds), form the natural reservoir for low pathogenic avian influenza (LPAI) viruses [1], [3]. In these birds, LPAI viruses are generally thought to cause asymptomatic infections. Avian influenza viruses can also infect numerous other species such as domestic birds, humans, pigs, horses, and marine mammals [1]. When LPAI viruses of the H5 and H7 subtypes are introduced into poultry, highly pathogenic avian influenza (HPAI) virus may emerge as the result of the acquisition of a multiple basic amino acid sequence at the HA0 cleavage site that allows systemic virus replication in poultry [4]. During numerous outbreaks in poultry, H7 viruses have been transmitted to humans, in which they frequently caused conjunctivitis and less frequently respiratory illnesses [5]. HPAI H5N1 viruses are still circulating in poultry and wild birds on the eastern hemisphere, predominantly in Asia, Africa, and the Middle East. HPAI H5N1 viruses have been transmitted to humans on numerous occasions, often resulting in severe disease, with a reported case fatality rate of about 60% [6], [7]. It is feared that the HPAI H5N1 virus could cause a pandemic if it would acquire the ability to transmit efficiently between humans. Since April 2009, H1N1 viruses of swine origin have been detected in humans, and caused the first pandemic of the 21st century [8], [9]. This pandemic and the numerous recent influenza virus zoonoses have highlighted the need for reagents and technologies for basic research and for the development of diagnostic tools and vaccines.

In response to the viral zoonoses and presumed pandemic threat(s), numerous large-scale surveillance studies have been initiated in wild and domestic birds around the globe, to screen for the presence of influenza viruses. Virus collections obtained from such studies performed in wild birds in Northern Europe [10], [11], [12], [13] were used here to establish a repository of plasmids and recombinant viruses representing all contemporary Eurasian HA and NA subtypes. Prototype strains were selected based on genetic data. The HA and NA genes of the respective strains were cloned in plasmids that can be used to rescue recombinant viruses with reverse genetics techniques or to express HA and NA proteins by transfection [14]. Recombinant viruses, consisting of six gene segments of influenza virus A/Puerto Rico/8/1934 (A/PR/8/34) and the HA and NA gene segments of avian viruses were generated. A/PR/8/34 was chosen because it is attenuated in humans and can therefore be used safely in laboratory settings without strict biosafety regulations [15]. Moreover, A/PR/8/34 is generally the strain of choice to generate vaccine seed strains, because of the relatively high yields of virus production in embryonated chicken eggs or mammalian cell cultures [16], [17]. Two sets of recombinant viruses were generated. The first set included all HA and NA subtypes, mostly in combinations as they were found in wild birds. This set may be useful to serve as reference reagents for the determination of influenza A virus subtypes in diagnostic settings [18], to serve as prototype vaccine candidates, as well as for basic research purposes, including structural and functional studies on HA and NA. The second set included recombinant viruses of the H5, H7, and H9 subtypes that are frequently associated with large-scale outbreaks in poultry, and that expressed heterologous NA genes of the N4 and N5 subtypes. N4 and N5 were chosen based on the very low prevalence of these NA subtypes in wild and domestic birds, and very low frequency of detection of these NA subtypes in combination with H5, H7, and H9 [11], [19]. As a result, the H5, H7, and H9 viruses with N4 and N5 NA genes may be useful as prototype poultry vaccine strains, to be used following the “Differentiating Infected from Vaccinated Animals (DIVA)” strategy [20].

Section snippets

Cells

293T cells were cultured in DMEM (Lonza, Breda, The Netherlands) supplemented with 10% FCS, 100 IU/ml penicillin, 100 μg/μl streptomycin, 2 mM glutamine, 1 mM sodiumpyruvate and, non-essential amino acids. Madin-Darby Canine Kidney (MDCK) cells were cultured in EMEM (Lonza) supplemented with 10% FCS, 100 IU/ml penicillin, 100 μg/μl streptomycin, 2 mM glutamine, 1.5 mg/ml sodiumbicarbonate, 10 mM Hepes, and non-essential amino acids.

Viruses

Most of the viruses used in this study were obtained from ongoing

Selection of viruses

To establish a repository of reverse genetics vectors containing all HA (H1–H16) and NA (N1–N9) subtypes, a selection was made from viruses isolated during influenza virus surveillance studies in wild aquatic birds in Northern Europe [10], [11]. Because viruses of subtypes H14 and H15 have not been detected in recent surveillance studies, older representative strains were used for these virus subtypes [12], [13]. To select representative strains, phylogenetic trees were generated that included

Discussion

The classical way to produce influenza virus vaccine seed strains is by reassortment of two viruses in embryonated chicken eggs, one virus with desired antigenic properties of HA and NA, and a second virus with a high yield phenotype, usually A/PR/8/34 [27]. Recent development of reverse genetics techniques facilitated the production of viruses containing the desired HA and NA subtype with any influenza virus backbone [14], [28], [29], [30]. Recombinant or reassortant viruses can subsequently

Acknowledgments

We thank Dr. Webster for the H14N5 and H15N9 viruses, Dr. Hoffmann for pHW2000, and Dr. Garcia Sastre for the pCAGGS plasmid. We thank C. Baas and P. Lexmond for excellent technical assistance and M. de Graaf for helpful discussions. This study was financially supported by ZonMW grant 91402008, awarded to the Netherlands Influenza Vaccine Research Centre, and National Institute of Allergy and Infectious Diseases–NIH contract HHSN266200700010C.

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    Present address: Laboratory of Virology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA.

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