Detecting Virulence Determinants Of H5N1 Avain Influenza Virus For Ducks

Wild waterfowl, including ducks are natural hosts of H5N1 influenza virus. Early strains rarely caused disease in natural hosts. The infected hosts showed no sign of disease, but could shed virus to enviroment which other annimals can be easily infected. However with the evolution of H5N1 virus, reemerging H5N1 influenza virus in HK in 2002 are highly pathogenic to wild migratory birds and resident waterfowl. Late in 2005 an outbreak of H5N1 virus infection occurred among migratory birds at Qinhai Lake in china. However the molecular basis for H5N1 virus transforming from low pathogenicty to higy pathogenicity for waterflow is still unkwon.The present studies characterised pathogenicity of 13 strains of H5N1 influenza viruses for ducks which islolated from china during 2001 to 2005. The results showed there are highly, moderately or low pathogenic for ducks. This indicated diverse pathogenicity among H5N1 viruses for ducks in china. So domestic ducks will still play an important role in epidemiology of H5N1 avian influenza virus in china. To understand the genetic determinant of virulence for duks, two viruses whose virulence differ a lot, but with higly similarity of their genome were selected as model viruses. DK/HB49 was highly pathogenic for ducks with its DLD50, 2.5lgEID50. All the infected duks were killed within 3-5 days p.i. with dosage of 106 EID50; On the contraty, GS/HB65 was low pathogenic for ducks with its DLD50, >8.5 lgEID50. All the infected ducks did not show any clinic signs. Comparing the virus titre level in different organs, it was found a distict titre differnce in brain of the ducks between incoluated with DK/HB49 and GS/HB65(107.6EID50/ml VS 102.5EID50/ml), when the ducks inoculated with dosage of 106 EID50. But no statistical titre difference present in lung, pancreas, and kidney. So we hypothesized both two type viruses (highly pathogenic and low pathogenic) could enter duck brain, whlie virulent virus could replicated efficiently in duck brain cells, but low pathogenic vrius cannot. That was main reason for causing infected ducks to death.Then we established reverse genetic system for DK/HB49 and GS/HB65, and rescued a series of reassortant and mutant viruses. Further these viruses were tested their virulence for ducks. At last we found PA gene contribute the virulence of these two viruses. The virulence of the reassortant bearing the PA gene from DK/HB49 in the GS/HB65 background was increased 105-fold relative to that of the GS/HB65 virus. Whlie at background of DK/HB49 only 65PA was substituted for 49PA, the pathogenicity of the reassortant virus was discreased by 102-fold. The virus titer in the brian of ducks infected with DK/HB49 was enhanced greatly compared with those ducks infected with GS/HB65. In order to pinpoint the key amino acids that affected virulence, we generated many PA mutant viruses and tested them on ducks. These results indicated the combination of the two amino acid mutations at positions 224 and 383 of PA protein determined the pathogenicity of GS/HB65 and DK/HB49.We have found the two amino acid mutations at positions 224 and 383 of PA involved in pathogenicity of these two viruses. What is the exact mechnism? With this question we performed mini-genome assay to detect the change of polymerase activity when mutant PA at positions 224 or /and 383 were introduced. It showed substitution of amino acid at position 383 changed the vrial polymerase dramatically both in 293T and in DF1 cells . The amino acid at position 383 in the PA protein also affects the transport of PA and PB1 into the nucleus of DF1 cells. Substitution of amino acid at position S224P of PA protein result in loss of an potential phsophorylation site . However, the mechanism underlying how the S224P mutation in the PA protein affects the virulence of the H5N1 virus in ducks remains to be determined. By the way substitution of amino acid at position 224 or/and 383 did not affect PA itself stablity and interation with DK-CLE.In summary, we compared two naturally isolated H5N1 avian influenza viruses that differ in their lethality in ducks, and demonstrated that the polymerase PA plays an important role in the increased virulence of the H5N1 DK/49 virus in ducks. We report for the first time that the two amino acids 224P and 383D in PA are critical and have cumulative effects on the highly lethal phenotype of the virus to ducks. The amino acid at position 338 in PA plays an important role in the activity of the polymerase and in the accumulation of the polymerase PA and PB1 subunits in the nucleus of virus-infected cells. Our results demonstrate that PA is a virulence factor of H5N1 avian influenza viruses.