Cloacal swabs were obtained from 8 chicken farms in Lagos (1), Ogun (5), Oyo (1) and Ekiti (1) States from June through November 2007. RNA extraction from cloacal swabs, reverse transcription–PCR amplification, and gene sequencing were conducted as described (1). For most viruses, complete sequences were obtained for all gene segments. Kimura distances were calculated on the basis of complete or partial gene sequences by including the maximum sequence length available from all strains included in the comparison. Phylogenetic trees were calculated by using PAUP version 4.0 beta 10 (7) with the maximum-likelihood method. The best model was determined by using MODELTEST (8). The sequences have been submitted to GenBank with the accession nos. FM160635–FM160642 and FM164800–FM164855.

Gene sequences of all 8 HPAI viruses (H5N1) described here were more closely related to sublineages A or C strains found in Nigeria than to any other published H5N1 virus subtypes. In particular, they were more closely related to the first strains found in Nigeria in the beginning of 2006 than to any strains found outside the country. Thus, the viruses detected in southwestern Nigeria during the second half of 2007 probably evolved from the first viruses brought into the country in early 2006 (1), suggesting that HPAI (H5N1) has continuously circulated and is endemic to Nigeria. Sublineage A viruses have continued to circulate in Nigeria, whereas sublineage B was found only once on 1 farm (SO layer farm, Lagos, January 2006), and sublineage C viruses were no longer detected in 2007. Sublineage A viruses have been detected in northeastern Nigeria in February 2007 (15) and in 2 states of southwestern Nigeria during the last quarter of 2007 (A/chicken/NIE/EKI15/2007 and A/chicken/NIE/OYO14/2007). Sublineages B and C viruses may have been eliminated in Nigeria by effective countermeasures.

All AC_HA/NS described here were obtained from chicken flocks in Ogun State from June through August 2007. These results are similar to those found in the beginning of 2007 in other states of Nigeria (15). In addition, we identified an AC_NS reassortant in Lagos State (A/chicken/NIE/LAG6/2007) distinct from the latter strain. At least 2 distinct reassortment events were necessary to generate sublineages A and C reassortants AC_HA/NS and AC_NS, which probably had occurred already in 2006, as suggested by the conspicuous absence of sublineage C in 2007. Although it is obviously more difficult to demonstrate reassortment events between genetically similar viruses, the asymmetry in gene divergence of A/chicken/NIE/OG5/2007 compared with the other AC_HA/NS and AC_NS reassortants suggests that additional reassortment events have taken place.

In 2006, only 1 reassorted strain was found among 35 European–Middle Eastern– African strains, including 19 viruses reported from Nigeria, belonging to 3 parent sublineages (1–4). In the beginning of 2007, 10 of 12 from northern, southern, and central states all belonged to the same AC_HA/NS reassortants (15), distinct from the AC_PB1/HA/NP/NS reassortant detected in 2006 (3). Similar reassortants were also found in other regions of sub-Saharan Africa (unpub. data). During the second half of 2007, we found 6 reassortants including 3 distinct reassortants among 8 strains collected from 8 farms located in 4 contiguous Federal States of Nigeria (Figure 2). These results suggest that reassortants have largely replaced the initial sublineages from which they were derived and that reassortments are pervasive. This finding confirms that reassortments between subtype H5N1 viruses occur frequently when different strains cocirculate in the same region (16) and is of particular concern if the increasing prevalence is the result of adaptation to the African environment.

Although segments of the replication complex (PB1, PB2, PA, and NP) may reassort individually without affecting viral fitness (16), there seems to be a coordinated evolution of the HA and NA genes (17). In all but 1 of the Nigerian reassortants, HA and NA genes originated from different sublineages (C and A), suggesting compatibility between phenotypes of both sublineages. All reassortants from Nigeria included sublineage C–derived NS genes, which may suggest a higher fitness of these viruses. Sublineage C–derived NS1 and NS2 sequences from all Nigerian reassortants and 11 unpublished sequences from AC_HA/NS reassortants identified in other sub-Saharan regions showed 2 amino acids (NS1 V194 and NS2 R34), which were never identified in sublineage A viruses. It has been shown that modifications in the NS proteins, including amino acids adjacent to V194, may modulate the virulence of HPAI (H5N1) (18,19). Alternatively, the observation that all reassortants in West Africa have sublineage C–derived NS genes may suggest a better adaptation to the African environment of viruses that came from the cold temperatures of central Asia. Thus, the influence of differences in ecology between Africa and Eurasia on viral selection and dynamics deserves further attention.

Although no reassortments have been reported among clade 2.2 viruses (www.who.int/csr/disease/influenza/tree_large.pdf) in Central Asia, Europe, and the Middle East since their emergence from Qinghai Lake region in 2005, reassortments of these viruses seem to be rampant in sub-Saharan Africa, where they have become the critical determinant of genetic diversity of HPAI (H5N1). Because of low prevalence, mainly in wild birds, clade 2.2 viruses have few opportunities to reassort in Eurasia. In contrast, opportunities to reassort seem to be frequent in sub-Saharan Africa because of great difficulties in setting up a sensitive surveillance system in a complex socioeconomic environment, where backyard farms and large commercial farms with variable biosafety levels coexist, and where culling may threaten the livelihood and survival of the farm.

If the high prevalence of reassortants was typical for West Africa in 2007, the absence of such reassortants anywhere else suggests that reintroductions of subtype H5N1 from Western Africa into Eurasia must be rare. Moreover, all HPAI (H5N1) strains from Nigeria in 2007 were more similar to those found in Nigeria in 2006 than to even the closest relative from Europe in 2007 (Hungary). Although subtype H5N1 has been found in wild birds from Africa, such as vultures (4), HPAI (H5N1) has so far not been reported in long-distance migrating birds in West Africa. Thus, the exchange of subtype H5N1 between Eurasia and Africa seems to be a rare event, which in 2006 may have been triggered by unusual bird migration as a result of the central Asian cold spell.

The biological significance of reassortments between genetically similar viruses may be arguable, but the frequency of reassortment events is an important marker of virus endemicity in a region. Moreover, endemicity of HPAI (H5N1) and a high propensity of reassorting in a region where seasonal influenza is unchecked are essential ingredients of the anticipated pandemic.