A/Vietnam/1203/04 (H5N1) virus isolated from a human and grown in chicken embryos produced a heterogeneous virus population that formed two types of plaques in MDCK cells, differing in size and in pathogenicity for ducks, ferrets, and mice (summarized in Table ). Large-plaque isolates, like the wild-type virus, were highly pathogenic in ducks, ferrets, and mice, whereas the small-plaque isolate was nonpathogenic in ducks, mice, and ferrets. Sequence analysis of the entire genome revealed five encoded amino acid differences between these two isolates. We utilized reverse genetics to investigate which residues in the large-plaque (high-pathogenicity) virus would convert the phenotype to low pathogenicity in ducks, mice, and ferrets.
Summary of mortality caused by A/Vietnam/1203/04 wild-type, plaque-purified, and reverse-genetic viruses after infection in different species
The rg construct of the A/Vietnam/1203/04 (H5N1) virus behaved essentially like the large-plaque virus. The rg wild-type construct killed fewer ducks than the original virus but made all ducks extremely ill and behaved phenotypically like the large-plaque isolate. The slight differences in mortality in the ducks probably reflect the biological heterogeneity of the outbred wild mallard ducks used in the studies.
Neither of the HA gene mutations (K52T or A544V), when inserted into the wild-type virus, markedly affected the properties of the rg viruses; neither change prevented mortality in mallard ducks or in mice. The HA2 construct (A544V) killed fewer ducks but was transmitted efficiently to contacts, and the birds had neurological disease and cloudy eyes. These results show that the HA gene is not individually responsible for the low pathogenicity of the small-plaque viruses in ducks and mice.
The mutations in the PA (T515A) and PB1 (Y436H) genes abolished the pathogenicity of the constructs in ducks. However, these constructs still replicated in ducks, were transmitted to contacts, and were able to kill ducks if inoculated intravenously. The latter finding indicates that an early step in infection was probably affected.
It is noteworthy that the virus with the Y436H mutation in PB1 was not transmitted as efficiently as other viruses in ducks; virus was detected late in the contact ducks (7 days postexposure), and virus was not detected in tracheal or cloacal samples from multiple inoculated and contact ducks. Therefore, the Y436H mutation in PB1 compromises transmissibility.
Pathogenicity studies in ferrets and mice were previously performed, comparing two different virus isolates from the same human patient (A/Vietnam/1203/04 [H5N1] and A/Vietnam/1204/04 [H5N1]). These two viruses differed by eight amino acids, including amino acid position 627 in the PB2 gene (K or E), yet both viruses were highly pathogenic in mice and ferrets (23
). These two isolates from the same patient also contained the R207K and H436Y differences in the PB1 gene that were found in the present study.
The A/Vietnam/1203/04 (H5N1) virus with a lysine at residue 627 of PB2 was 40-fold more virulent in mice than the A/Vietnam/1204/04 (H5N1) virus that possesses a Glu at this residue. All of the viruses used in this study had a lysine at residue 627 of the PB2 gene. While the A/Vietnam/1203/04 (H5N1) strain containing the Lys627 was slightly more pathogenic in mice, both isolates (possessing either Glu or Lys at residue 627) were highly pathogenic in ferrets. Thus, the molecular requirements for high viral pathogenicity differ depending on the host.
It is noteworthy that a single amino acid change in the PA (T515A) gene converted a lethal virus to a nonlethal virus in ducks but retained high pathogenicity for mice and ferrets. Little is known about the molecular basis of transmissibility, but the PB1 Y436H change appears to have made the virus less fit. However, the small-plaque variant itself was transmitted efficiently in ducks, suggesting that other molecular interactions can overcome the problem.
Although the changes in the PA (T515A) and PB1 (Y436H) genes reduced lethality in ducks inoculated by natural routes, both of these viruses killed ducks when they were injected intravenously. This finding indicates differences in compatibility of replicative precursors in cells available at the surface of the upper respiratory and digestive tracts and after systemic exposure by intravenous injection. Thus, host cell tropism contributes to the complexity of pathogenesis.
Although we found that differences in lethality in ducks were associated with differences in the PA (T515A) and PB1 (Y436H) genes, we did not identify the mechanism involved. Our evidence suggests that the viruses with these changes replicate to the same levels in the same organs. Thus, the wild-type virus, the small-plaque isolate, and the large-plaque isolate had indistinguishable replication levels both in ducks and in ferrets. Possible explanations are (i) that the differences are immunological and cytokine based or (ii) that these are differences in polymerase activity and that additional testing is needed to determine whether it takes longer for the virus to get to different tissues.
The association of high pathogenicity in ducks with the polymerase genes (PA and PB1) is in keeping with earlier findings that the polymerase genes contain important determinants of pathogenicity (9
). However, the residues involved differ between hosts. As mentioned above, residue 627 of PB2 is important in determining pathogenicity in mice but not in ferrets. Here we have shown that a single change in the PA gene (T→A) reduces pathogenicity for ducks but not for mice or ferrets. It is clear that high pathogenicity involves a complex of viral and host genes, but in most cases, the polymerase genes in concert with the NS and HA genes play a role; the connection between this role and the type of host is unresolved.
While H5N1 viruses are highly pathogenic in some duck species (e.g., tufted ducks), the highly pathogenic phenotype appears to be selected against in others, such as the mallard and the grazing ducks of Thailand and Vietnam. Highly pathogenic avian influenza viruses (H5 and H7) must over the centuries have spread to ducks, yet the influenza virus phenotype of high pathogenicity has not previously been perpetuated in wild aquatic birds. It seems unlikely that in the long term, HP H5N1 viruses will be perpetuated in the aquatic bird reservoir. However, the continuing isolation of HP H5N1 viruses from domestic ducks since 1996 is of concern, as the domestic duck (3
) may perpetuate H5N1 virus that is nonpathogenic in these ducks but that remains pathogenic for other species.
The present study provides the first insight into the high pathogenicity of H5N1 viruses in ducks, but much remains to be done. An important unresolved question is whether the HP H5N1 virus is currently perpetuated in migrating or in domestic waterfowl. Detailed prospective surveillance of both domestic and wild waterfowl is needed to answer this question.