Sequence Analysis of California and Western Avian Influenza Virus Isolates

Non-Technical Summary- Avian influenza is one of the most important diseases of poultry. The highly pathogenic form is an OIE List A disease and is rapidly fatal in naive poultry populations. In addition, the recent demonstrations of the zoonotic potential of this virus add to its importance. Few avian influenza isolates from California and the Western region have been characterized or even partially sequenced. This project will provide sequence data on the historical avian influenza isolates from California and the Western US. This sequence data will serve as background information on AIV in the West.
<p> Approach- We have selected fifteen avian influenza viruses (AIV) isolated in California from poultry in California and in the Western US. They include isolates from duck, quail, turkey, chicken and emu. They include viruses of the following subtypes: H10N7, H9N2, H6N2, H5N2, and H11N9 among others. All of the selected isolates are non-pathogenic or of low pathogenicity. These viruses will be expanded in embryonating SPF eggs using standard methods (Woolcock et al., 2001). Avian influenza virus RNA will be extracted from the allantoic fluid using the RNeasy kit (Qiagen, Valencia, CA). The purified RNA will be reverse transcribed using random hexamers (ABI). Universal primers will be used to amplify each of the eight genetic segments of each virus (Hoffman et al., 2001). These genetic elements will be cloned using the TopoTA cloning system (Invitrogen, Carlsbad, CA). We will sequence the complete HA and NA genes and we will sequence 400 bases each of the remaining six genes. We have selected to clone all genes in order to stabily store them for future use in other projects. We will only sequence 400 bases of each isolate at this time because that is all we need in order to 1) determine the lineage of the gene segment and 2) potential relatedness to other isolates in the Influenza Sequence database and/or GenBank. Sequencing of the cloned genes will be performed in both directions and contiguous sequences constructed using the ContigExpress program (Vector NTI, 9.0, Informax) by a sequencing service (Davis Sequencing, Davis, CA).