[Biological significance of amino acids deletion in NA stalk of H5N1 avian influenza virus].

It has been reported that NA gene of some H1N1 Influenza A virus strains isolated since 1933 is characterized by a deletion of 11 to 16 amino acids in the stalk. The spontaneous mutant in NA stalk of H1N1 virus lacks enzyme activity with large substrate (fetuin) but not with small substrate (sialyllactose). Recently, H5N1 virus also has been found that NA has the same unique mutation in the stalk, a deletion of 15 to 20 amino acids. However, biological significance of this mutation has not yet been reported. In order to investigate biological significance of the amino acids deletion in NA stalk of H5N1, five reassorted H5N1/PR8 viruses were generated via eight-plasmid based reverse genetics system. These five viruses were named 506, m506-, 646, m646+ and 196, respectively. The six internal genes of recombinants were all from A/PR8/34(H1N1), and HA gene was from A/G/JS/03(H5N1), however, they had different NA genes. 506 and m506- held NA fragments derived from A/G/HD/00(H5N1), and the former was distinguished with a longer NA which had no 20 amino acids deletion in the stalk. 646 and m646+ held NA fragments from A/G/JS/03(H5N1), and the NA stalk of m646+ was 20 amino acids longer than that of 646. The NA of 196 was derived from A/PR8/34 which had 15 amino acids deletion in its stalk. Biological characteristics of these viruses showed that recombinants with different NA length could grow well in embryonated SPF eggs, and their EID50, MDT, and viral titers were similar. However, the length of NA was related to the capacity of eluting viruses from erythrocytes for 506 and 646+ which holding longer NA stalks eluted from erythrocytes more quickly than m506-, 646 and 196 did. Moreover, 15 or 20 amino acids deletion in NA stalk had a pronounced effect on virus growth ability in MDCK cells. Viral titers in supernatant of MDCK infected with m506- or 646 were 10 to 100 folds higher than those infected by 506 or m646+. And the plaque size of m506- and 646 were larger than that of 506 and m646+. The results reveals that H5N1 AIV with amino acids deletion in NA stalk would expand its host range. The unique amino acids deletion in NA molecule of H5N1 may be associated with the adaptation of virus to terrestrial poultry or the increasing ability of interspecies transmission.

[Establishment of reverse genetics system for H5N1 subtype AI].

H5N1 subtype influenza virus A/Duck/Shandong/093/2004 (A/SD/04) strain was chosen as the master strain for rescue research. 11 sets of primers for 8 plasmids construction were designed base on the sequencing of the full-length of A/SD/04. Eleven fragments of A/SD/04 were amplified by the designed primers and were ligated with PHW2000 for rescue plasmid construction. Eight transcription/expression plasmids were obtained, which encoded the eight segments of A/SD/04, and designated as 241, 242, 243, 244, 245, 246, 247 and 248, respectively. The COS-1 cell was cotransfected with eight plasmids with different combination of A/SD/04 and PR8. The eight reassortants shared the same HA (from A/SD/04) but contained different internal genes and NA. All of the eight reassorted viruses had some similar bio-characteristics, such as the viral title in fertilized eggs was range from 256 to 1024, the EID50 were between 10(-8.5) to approximately 10(-9), and MDT were between 34 to approximately 46h. But the IVPI of the eight reassortants was differently and all were lower than the wild-type A/SD/04. These results confirmed that different recombination of internal genes of H5N1 has influence on viral virulence to 6-week SPF chicken but not on viral replication ability in embryonated chicken eggs. The establishment of eight-plasmid rescue system for A/SD/04 is the base for farther research on genes function of H5N1. And A/SD/04 can be used as a backbone to replace PR8 entirely in generation of H5 AIV vaccine candidate.

[Cloning of full-length genes of H5N1 subtype Avian influenza virus strain A/duck/Shandong/093/2004 and analysis of the sequences].

The eight full-length genes, including the 5' and 3' ends of H5N1 subtype Avian influenza virus (A/duck/ Shandong/093/2004) were amplified by using the universal primers and H5 specific primers. The method used for the amplification of Avian influenza virus's full-length sequence was more easily and rapidly than that of rapid amplification of cDNA ends assay (RACE). The amplified segments were cloned into the T vector PCR 2.1, respectively. Three to five positive clones of each gene were sequenced and the same two sequencing results of the full-length genes were obtained. The phylogenetic analysis results showed that all the eight segments of the A/duck/Shandong/093/2004 were different from the A/Quail/Hongkong/G1/97 and A/Chicken/Beijing/1/94, but showed highly similarity (99% and above) to that of four H5N1 strains, which were isolated in 2002 in duck. It revealed that this strain was resulted from re-assortment of H5N1 rather than H9N2. The NA sequence of A/D/SD/04 was analyzed and the result demonstrated that there are 20 amino acids missing in 48 - 68 sites, however, there was no residue lost in NS gene in 263 to 277 sites. The motif of HA cleavage site is PQRERRRKKR/G, which is the characteristic of HPAIV. The 226 amino acid residue was Met (M), which can react with both Aalpha-2, 3Gal and SAalpha-2, 6Gal receptor. And the 627 residue of PB2 was Glutamic acid (E). The result mentioned above confirmed that H5N1 subtype AIV has multiple determinants in its virulence. A/D/SD/04 is the mid-strain evolving from HPAIV to a virulent strain of mammal.