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H5N1 Bird Flu Recombination in China and Vietnam
Recombinomics Commentary
July 20, 2005
In our studies, ducks that survived experimental infection with highly pathogenic H5N1 influenza virus shed virus for a prolonged period. Virus that was shed during the latter part of this period was nonpathogenic to ducks and was antigenically distinguishable from the input viruses; however, these viruses remained highly pathogenic to chickens.
Sequence analysis of the HA confirmed that variants with amino acid changes had been selected. Analysis of the highly pathogenic avian H5N1 virus samples used to infect the ducks suggested the presence of a mixed virus population. Sequence analysis of the original material showed multiple double base peaks, further supporting the presence of mixed populations.......
In this study, we see virus shedding for up to 17 days after infection with the 2003-2004 H5N1 virus isolates.Therefore, it appears that the viruses that have emerged since 2002 have viral characteristics that have changed to a great extent compared with viruses isolated before 2002.
The cloacal shedding of virus observed in both pathogenicity groups indicates that long-term shedding is not infrequent or confined to one pathogenicity group. This characteristic is of great consequence especially in free-ranging ducks in that it increases the likelihood of transmission of virus to the environment, to other ducks, and, potentially, to other species. Water in which ducks swim, drink, and eat presents a high exposure risk to humans and domestic chickens. The risk is greatest in the rural areas of affected countries, where domestic ducks and chickens often mingle, frequently sharing the same water supply. The viruses are potentially transmitted to chickens under these conditions.
Two different plaque-purified viruses from the same original sample had different pathogenicity profiles in mallard ducks. As the virus replicates in the host, the predominant virus is targeted by host antibodies and is largely eliminated by the immune response. The minor virus population is then able to replicate and is shed at detectable titers over an extended period.
The above discussion in yesterday's PNAS provides additional evidence for dual infections and offers additional support for the WHO warning last October about the spread of H5N1 bird flu by asymptomatic ducks.
The article mentions changes in sequences in birds infected with two or more H5N1 viruses. These changes are due to recombination, which is driving the evolution of H5N1. The evolution is rapid, because the number of dual infections is increasing because of the large numbers of birds infected. H5N1 is also acquiring mammalian sequences, which is expanding its host range.
The acquisition of mammalian sequences can happen in swine and people, which is why the mild cases in northern Vietnam and throughout China can accelerate this change. As mammalian polymophisms are accumulated, more recombination can happen via the increase in the regions that provide exact matches. Thus the evolution is not linear, but can accelerate dramatically.
The acquisition of small regions is clear from new sequence data. The 2005 H5N1 sequences at Los Alamos show that almost all isolates have lost the ARG residue in the HA cleavage site. This is not a new mutation, but acquisition of a mutation previously circulating in China, Japan, and South Korea in 2003 and 2004. Similarly, the new isolates contain polymorphisms that match polymorhisms seen in Yunnan previously. The isolates from Qinghai have the human PB2 polymorphism E627K. This polymorphism is found in all human flu isolates, from 1993 to present. In avian H5N1 isolates, this change was only seen in avian isolates passed through mouse brains. Now it is all H5N1 Qinghai isolates.
Boxun reports indicate two additional strains L33 and RW4 are circulating in birds in the Qinghai Lake area in addition to RW4 (which would be equivalent to the virulent strain described in Nature and Science). The cocirculation of these three strains is cause for additional concern because they can reassort and recombine to create additional versions of H5N1 and these new versions will be flying to India, eastern China, and Europe in the next few weeks. Moreover, boxun reports details on human cases in the Qinghai Lake area and China is not responding to a WHO request to visit Xinjiang or to provide additional samples and sequences from Qingahi or Xinjiang.
These data strongly suggest H5N1 is rapidly evolving, increasing its host range, and being covered up in China.
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