Introduction into Nigeria of a Distinct Genotype of Avian Influenza Virus (H5N1)

Genetic characterization of highly pathogenic avian influenza viruses (H5N1) isolated in July 2008 in Nigeria indicates that a distinct genotype, never before detected in Africa, reached the continent. Phylogenetic analysis showed that the viruses are genetically closely related to European and Middle Eastern influenza A (H5N1) isolates detected in 2007.

I n February 2006, highly pathogenic avian infl uenza (HPAI) virus of the H5N1 subtype was detected in chickens in Kaduna state in northern Nigeria, the fi rst African country reporting a confi rmed HPAI (H5N1) outbreak. The infection later spread to 25 of the 36 Nigerian states and to the Federal Capital Territory and persisted for 21 months. Consequently, ≈368,000 domestic birds (mostly chickens; also guinea fowl, turkeys, ducks, geese, and ostriches) were killed by the virus or culled, and a fatal human case was reported. By the end of 2007, the outbreaks appeared to have been controlled by such measures as stamping-out with compensation, restricting movement of poultry and poultry products, improving biosecurity measures, and enhanced surveillance systems. The last reported case of HPAI occurred in the southern state of Anambra and was reported by the Government of Nigeria in October 2007. In addition to the routine avian infl uenza surveillance program, industrial poultry in the 36 states plus the Federal Capital Territory, and live bird markets have been actively monitored since March 2007, with >13,000 samples collected and analyzed for avian infl uenza viruses (T. Joannis, pers. comm.).
During the surveillance activities at the live bird markets, new cases of HPAI (H5N1) were detected in July 2008 in the city of Gombe in the northeastern state of Gombe after a 9-month period during which no infl uenza A virus was identifi ed. In particular, 2 tracheal swabs collected from apparently healthy domestic ducks were submitted to the laboratory for virus isolation in embryonated specifi c antibody-negative fowl eggs. Allantoic fl uid harvested from inoculated eggs showing embryo death tested positive for hemagglutinating agents. RNA extracted from positive allantoic fl uid also tested positive by real-time reverse transcription-PCR for type A infl uenza RNA (1) and for the H5 subtype (2). Hemagglutination and neuraminidase (NA) inhibition assays with monospecifi c antiserum (3) confi rmed the H5N1 subtype. Viruses were designated as A/duck/Nigeria/3724-2/2008 and A/duck/Nigeria/ 3724-10/2008.

The Study
We obtained the full-length genome sequence for A/ duck/Nigeria/3724-2/2008 and the sequence of the hemagglutinin (HA) segment for A/duck/Nigeria/3724-10/ 2008 (4). Sequences of the 8 gene segments of A/duck/ Nigeria/3724-2/2008 were submitted to the Global Initiative on Sharing Avian Infl uenza Data public database (accession nos. EPI161701-EPI161708). The HA segment of the 2 isolates was identical, and the deduced amino acid sequence of the HA cleavage site was characteristic of HPAI (H5N1) (PQGERRRKKR*GLF). The highly pathogenic pathotype was confi rmed by the result of the intravenous virus pathogenicity index test (index 2.87) (3).
Phylogenetic analysis of the 8 genes was conducted by using MEGA 4 (5) with the neighbor-joining method, and the HA and NA tree topology was confi rmed by using Bayesian methods (6) (Figures 1, 2). Phylogenetic analysis of the HA gene segment showed the viruses fall in clade 2.2, according to the unifi ed nomenclature system (7). Unexpectedly, the viruses were grouped separately from the viruses previously detected in Nigeria and in other African countries. They clustered in the sublineage here designated III, together with HPAI (H5N1) viruses isolated in 2007 in Europe and Middle East (Figure 1). Phylogenetic analysis of the NA gene segment of A/duck/Nigeria/3724-2/2008 supported these results ( Figure 2 (8) or mutations related to resistance to NA inhibitors and to adamantanes were observed in the HA, NA, and M2 genes of the Nigerian isolate. Analysis of the amino acid sequences of the internal proteins of A/ duck/Nigeria/3724-2/2008 virus showed the amino acid lysine at position 627 of the PB2 gene known to be associated with increased virulence of HPAI (H5N1) virus in mice (9) and 1 amino acid mutation at position 33 (V33I) of the NP gene, which is described as genetic signature of human infl uenza A virus (10). The PB1-F2 protein had 1 mutation at position 66 (N66S), previously observed only in the Hong Kong 1997 subtype H5N1 viruses and in the 1918 pandemic strain (A/Brevig Mission/18) and is associated to high pathogenic phenotype in mice (11).

Conclusions
Since the earliest known progenitor detected in China, A/goose/Guandong/96, numerous lineages of HPAI (H5N1) viruses have emerged (7). Introduction and spread of distinct H5N1 genetic lineages were described in several Asian countries and in Europe, such as Germany, Italy, and France (12,13). Surprisingly, Nigeria is the only country in Africa where HPAI (H5N1) belonging to distinct sublineages have been detected (4,12,14). Previous genetic characterization of HPAI (H5N1) viruses isolated during 2006 and 2007 indicated the cocirculation in Nigeria of 3 distinct sublineages, here designated I, II, and IV (4,14). Sublineages I and II appeared to be widespread in this country during 2006 and 2007 (14), and their extended cocirculation enabled reassortment events between these sublineages. The fi rst reassortant virus was identifi ed in June 2006 (12), and in early 2007, an additional reassortant virus was identifi ed in 7 of 22 Nigerian states where infection was found (4).
Our results indicate a novel introduction in Nigeria of a virus belonging to sublineage III, a genotype not previously detected in the African continent. Indeed, previous surveil-   (15). This fi nding, however, does not shed light on how the virus was introduced into Nigeria because neither of the 2 main means of spread, through wild birds or the poultry trade, can be excluded.
The evidence of a mutation in the PB1-F2 gene segment that increases the pathogenicity of the virus in mammalian hosts is of concern. This type of mutation has never been observed in HPAI (H5N1) viruses of clade 2.2; this fi nding supports the need for a continuous monitoring effort of infl uenza viruses A (H5N1) viral genotypes and their evolution. Our fi ndings highlight the evolving epidemiology of HPAI (H5N1) viruses and the need for implementation and maintenance of sustainable surveillance programs in countries where infection has been found and in countries where it has not. The outcome of these efforts, however, can be maximized only through prompt reporting to international organizations and international collaboration that leads to timely molecular and antigenic comparisons of isolates.