Reemerging H5N1 influenza viruses in Hong Kong in 2002 are highly pathogenic to ducks

Abstract

Waterfowl are the natural reservoir of all influenza A viruses, which are usually nonpathogenic in wild aquatic birds. However, in late 2002, outbreaks of highly pathogenic H5N1 influenza virus caused deaths among wild migratory birds and resident waterfowl, including ducks, in two Hong Kong parks. In February 2003, an avian H5N1 virus closely related to one of these viruses was isolated from two humans with acute respiratory distress, one of whom died. Antigenic analysis of the new avian isolates showed a reactivity pattern different from that of H5N1 viruses isolated in 1997 and 2001. This finding suggests that significant antigenic variation has recently occurred among H5N1 viruses. We inoculated mallards with antigenically different H5N1 influenza viruses isolated between 1997 and 2003. The new 2002 avian isolates caused systemic infection in the ducks, with high virus titers and pathology in multiple organs, particularly the brain. Ducks developed acute disease, including severe neurological dysfunction and death. Virus was also isolated at high titers from the birds' drinking water and from contact birds, demonstrating efficient transmission. In contrast, H5N1 isolates from 1997 and 2001 were not consistently transmitted efficiently among ducks and did not cause significant disease. Despite a high level of genomic homology, the human isolate showed striking biological differences from its avian homologue in a duck model. This is the first reported case of lethal influenza virus infection in wild aquatic birds since 1961.

FIG. 1.

H5N1 avian influenza virus outbreak in Kowloon Park, Hong Kong. Shown are the numbers of birds identified as sick or dead on each calendar day. H5N1 influenza virus infection was confirmed by virus isolation from the affected birds.

FIG. 2.

Survival (A) and weight loss (B) of mallards infected with different H5N1 influenza virus isolates. Ducks were observed for 7 days after inoculation with 1.0 ml of a 1:10 dilution of stock virus. Ducks with severe neurological signs were euthanized and were categorized as having died of infection. (A) Solid squares represent 8 of the 12 viruses tested, which did not cause mortality and whose lines overlap at the 100% survival rate. (B) Data points represent median values, and error bars represent the data range at each time point.

FIG. 3.

Virus titers in ducks experimentally infected with Gs/HK/739.2/02, in contact ducks, and in the drinking water provided for the ducks. Values plotted are the mean virus titers of all live birds on the indicated day (see Table 4). For calculation of the mean, influenza virus-positive samples with a virus titer of <1 log₁₀ EID₅₀/ml were assigned a value of 0.5 log₁₀ EID₅₀/ml. Index ducks (n = 3) were inoculated with 2 × 10^7.75 EID₅₀ of virus, and uninoculated contact ducks (n = 3) shared their cage, food, and drinking water.

FIG. 4.

Hematoxylin- and eosin-stained sections of the brain of a duck infected with Gs/HK/739.2/02. The brain was collected 6 days after inoculation with 2 × 10^7.75 EID₅₀ of virus. (A) A prominent inflammatory cell infiltrate is apparent in the meninges (*), neuropil (n), and perivascular spaces (arrows); the pallor of the neuropil indicates edema. (B) The inflammatory infiltrate in the neuropil and perivascular spaces comprises predominantly mononuclear cells. Magnifications: ×4 (A) and ×20 (B).