Progressive truncation of the Non-Structural 1 gene of H7N1 avian influenza viruses following extensive circulation in poultry.

In order to support eradication efforts of avian influenza (AI) infections in poultry, the implementation of "DIVA" vaccination strategies, enabling the Differentiation of Infected from Vaccinated Animals have been recommended by international organisations. A system, based on the detection of antibodies to the Non-Structural 1 (NS1) protein of AI has been proposed but the success of such a system lies in the conservation of the NS1 protein among different AI isolates. With this in mind, the ns1 gene of 40 influenza A viruses isolated from a spectrum of avian species was sequenced and compared phylogenetically. The isolates included both low pathogenicity (LPAI) (n=22) and highly pathogenic (HPAI) (n=18) viruses of the H7 subtype and were representative of the avian influenza viruses that circulated in Northern Italy from 1999 to 2003. Size variation in the predicted amino acid sequence of each NS1 was revealed with two different levels of carboxy-terminal truncation being observed. Of the 40 isolates analysed, 16 had a full-length NS1 protein of 230 aa, 6 had a truncated protein of 220 aa and 18 had an intermediate truncation resulting in a protein of 224 aa. All of the H7N1 HPAI isolates possessed the intermediate carboxy-terminal truncation. In addition, all of the H7N1 LPAI viruses circulating at the beginning of the epidemic had a full length NS1 while those circulating towards the end of the period had a truncated protein. To determine whether modifications to NS1 could be a result of laboratory manipulation, two strains (A/ty/Italy/977/99 and A/ck/Italy/1082/99) with a full length NS1 were inoculated into 10-day-old embryonated chicken and 12-day-old embryonated turkey eggs via the allantoic route for 20 blind passages and sequenced at passages 3, 10, and 20. No truncation was observed following these serial passages. To determine whether the truncation involved an immunogenic region of the NS1 protein a peptide spanning residues 219 aa to 230 aa was synthesized and tested in an indirect ELISA against sera obtained from turkeys experimentally infected with a virus strain known to have a full length NS1 protein. The peptide proved to be immunogenic highlighting the fact that the variations of the NS1 protein presented in this work must to be taken into consideration when developing a diagnostic test based on the identification of antibodies to the NS1 protein.