The emergence of H5N1 highly pathogenic avian influenza (HPAI) virus since it appeared in Hong Kong in 1997 (Claas et al. 1998
) has resulted in the exceptional spread of the virus to now cover over 60 countries spreading from China and the eastern regions of Russia in the east, through to Burkina Faso in West Africa and Scotland in northwest Europe. The extent of the spread of the H5N1 virus seems unprecedented and has not been seen previously in the second half of the twentieth century: in all other outbreaks of HPAI since the 1950s, control by culling and sanitary procedures usually resulted in the rapid containment and elimination of the virus. Culling, sanitary control measures and vaccination have been insufficient to contain H5N1 infection, although, for example, in Japan, South Korea and several European Union member states (OIE 2009
), early detection and culling of infected poultry proved effective in controlling HPAI H5N1 influenza.
The widespread dissemination of H5N1 viruses in poultry has resulted in a number of human infections and has been recognized to pose a threat of a human influenza pandemic. At the end of March 2009, over 410 human cases of infection had been confirmed, with a case-fatality rate of over 60 per cent. Most of the human cases have been associated with exposure to sick poultry, and there have been only few clusters of infection where human-to-human transmission cannot be ruled out (WHO 2009
). Human cases have been reported in 15 countries, with Indonesia, Vietnam, Egypt and China bearing the largest burden of human disease, accounting for 75 per cent of the cases. With the exception of Indonesia, human H5N1 influenza cases in each of these countries have been confirmed in the first three months of 2009. At the time of writing, the pandemic threat was defined by WHO as Phase 3, in which human infection is recognized from a new subtype but human-to-human transmission is rare. Should the rate of clusters of human-to-human transmission increase, then the pandemic risk phase will be increased (WHO 2005
It is striking that control of disease in animals shows an impact in the reduction of human infections. This is illustrated by the events observed in Vietnam between 2004 and 2007, where high numbers of human cases were seen in both 2004 and 2005 but dropped to zero in 2006, following the initiation of a vaccination campaign of poultry in 2005 when 160 million of an estimated population of 250 million poultry were vaccinated. The numbers of human cases rose somewhat in the following 2 years, but remain at a much lower level than that reported in 2004 and 2005 in the presence of continued poultry vaccination.
The reasons for the failure to eliminate H5N1 virus in domesticated poultry are complex but are likely to be associated with the widespread distribution of the virus before its presence is recognized and sanitary measures become enforced. A major factor that hampers control measures is likely to be the maintenance of highly pathogenic H5N1 viruses in different avian host species, a factor previously thought to be uncommon with other HPAI viruses. H5N1 infections have been observed in a diverse range of avian species: an estimate of the range of species infected with H5N1 viruses can be made from a survey of the genetically characterized viruses whose sequences have been made publicly available (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html
). As of March 2009, with over 2300 H5N1 haemagglutinin (HA) genes of avian origin analysed, approximately 1000 were from chickens, 650 from ducks, 200 from geese and around 50 each from swans and turkeys. These numbers can only be used as a very rough estimate of prevalence of the virus since domestic species have been under closer surveillance than wild birds in which infections are more difficult to detect. It is notable that species not normally associated with avian influenza virus infection have been found to be infected, and examples include sparrows, crows, magpies and storks, and birds of prey that may have fed on infected poultry, e.g. falcons (Monne et al. 2008
), kestrels (Smith et al. 2009
), vultures (Ducatez et al
) and buzzards (Hars et al. 2008
). The presence of H5N1 viruses in the bar-headed goose population around Qinghai Lake in late 2005 (Chen et al. 2005
) provides a striking example of wild-bird infection, and it is postulated that virus was carried from Qinghai Lake westwards through bird migration. The observations that, following experimental infection, a small number of ducks of several species showed severe disease signs lend support to the notion that spread through wild birds is feasible (Hulse-Post et al. 2005
; Sturm-Ramirez et al. 2005
; Keawcharoen et al. 2008
; Kim et al. 2008
; Londt et al. 2008
), yet the relative importance of wild birds in transmission of the H5N1 virus remains an open question.
The prevalence of H5N1 in wild birds has been studied intensively in Europe since 2006, and the results of a 2006 survey have been reported recently (Hesterberg et al. 2009
). The survey showed that H5N1 viruses were found only rarely in dabbling ducks, but swans, diving ducks, mergansers and grebes showed higher rates of infection and usually associated with disease. The survey, of over 120 000 birds sampled in 2006 with only 591 detected as positive for H5N1, concluded that this virus was not able to be sustained in the wild-bird population within the European Union. The continuing spread of infection seems to be very complex, and Yasué et al. (2006)
highlighted several important points in a review of the evidence for transmission of virus by wild birds over large distances. For example, in Qinghai Lake, bar-headed geese were not the only wild-bird species affected and there were subsequent reports of domesticated geese in the area. Also, which host infected the bar-headed goose population? And when were the geese first affected by H5N1 influenza? Yasué et al. (2006)
pointed out that bar-headed geese overwinter in India and arrive following their trans-Himalayan migration at the lake in March. The signs of H5N1 infection were first recorded around Qinghai Lake in May to July. It may be pertinent to note that Vijaykrishna et al. (2008)
estimate that the H5N1 virus was introduced into Indonesia and Vietnam three to six months prior to the first recognition of H5N1 infection in each country, so there may be a considerable delay in some areas between the index infection and the recognition of infection. Wild-bird migration has been proposed to be associated with the trans-African spread of H5N1 to Western Africa. Ducatez et al. (2006)
adduced that, in the emergence of virus in Nigeria, three separate introductions of the H5N1 virus coincided with the flight paths of migratory birds; nevertheless, commercial links are also known to exist between the poultry industries of Nigeria and the Far East, and the possibility of infected poultry importation or associated contaminated products cannot be excluded. The relative importance of the role of migratory birds in spreading H5N1 virus compared with the spread through human agricultural and food production activities needs careful consideration. It seems prudent to focus on the domesticated duck and poultry sector for the detection of H5N1 infection and for the control of the infection once found.
The widespread geographical distribution of H5N1 viruses, possibly combined with circulation in wild-bird species, has led to a wide diversity of H5N1 viruses. Phylogenetic analyses of the HA genes of H5N1 viruses have led to the construction of 10 clades defined according to phylogenetic criteria. However, as the number of genes sequenced has increased and as the virus continues to evolve, the initial phylogenetic classification of 10 clades has become more elaborate with the division of some clades into subclades and third-order clades (WHO/OIE/FAO Working Group 2008
The rapid evolution of the virus into the 10 recognized clades is likely to be associated with the dependence of the virus on an RNA-dependent RNA polymerase for replication. RNA viruses are considered subject to more sequence variation than viruses with less error-prone DNA-dependent DNA polymerases. There has been a long debate about whether RNA viruses exist as a virus quasi-species (e.g. Smith et al. 1997
; Holmes & Moya 2002
; Moya et al. 2004
) or whether they behave in a manner of standard population genetics, with selection at the individual virus rather than selection at the level of the virus population, a feature of a quasi-species. It has been pointed out many times (reviewed in the aforementioned references) that sequence variability does not imply quasi-species behaviour per se
. RNA viruses are thought of as having large populations, but they may undergo severe bottlenecks in transmission between hosts of the same, or different, species and result in a small effective population size (Moya et al. 2004
); it is well established in classical population biology that bottlenecks increase the fixation of neutral mutation (Maynard Smith 1989
) and hence virus population dynamics will influence the frequency of the fixation of mutations.
In the work we describe below, as part of a more detailed study into host selection of variant viruses, we investigated whether different avian influenza viruses showed any variation in the degree of diversity from the consensus sequence of the virus as they replicated in different hosts. We examined viruses of different natural history: a clade 2.2 H5N1 virus isolated from turkeys during the early phase of the introduction of this clade into Europe; an H5N1 virus, also of Eurasian origin, that represented a virus present prior to the explosive spread of the current H5N1 viruses and three H7N1 subtype viruses from an outbreak of avian influenza in Italy in 1999–2000. These viruses were used to infect groups of chickens, turkeys and ducks, and the variation in a region of approximately 1000 nucleotides in the virus HA gene was examined. In comparison, we also examined a smaller number of clones in a second RNA segment encoding the NS1 and NS2 (NEP) polypeptides of the virus; this gene was chosen as it underwent adaptive changes as the H7N1 epidemic emerged in Italy (Dundon et al. 2006
) and showed considerable reassortment and change as the H5N1 panzootic emerged (Li et al. 2004
; Duan et al. 2008