Molecular epidemiology of clade 1 influenza A viruses (H5N1), southern Indochina peninsula, 2004-2007

Abstract

To determine the origin of influenza A virus (H5N1) epizootics in Cambodia, we used maximum-likelihood and Bayesian methods to analyze the genetic sequences of subtype H5N1 strains from Cambodia and neighboring areas. Poultry movements, rather than repeated reintroduction of subtype H5N1 viruses by wild birds, appear to explain virus circulation and perpetuation.

Figure 1

Phylogenetic relationships of the hemagluttinin (HA) (A) and neuraminidase (NA) (B) genes of 33 Cambodian strains and of representative influenza A viruses (H5N1). Trees were generated by Bayesian analysis using MrBayes v3.1 software (2). Numbers above and below branches indicate Bayesian posterior probability and maximum likelihood bootstrap values (PHYML v2.4 software, www.atgc-montpellier.fr/phyml), respectively. Analysis was based on nucleotides 28–1578 of the HA gene and 67 to 1248 of the NA gene. Both trees were rooted to A/goose/Guangdong/6/96. Scale bar indicates 0.3 and 0.2 substitutions per site for HA and NA genes, respectively. Dk, duck; Ck, chicken; Gs, goose; Bhg, bar headed goose; Pg, pigeon; Gh, gray heron; Ql, quail; Mall, Mallard duck; Tg, tiger; CAM, Cambodia, VNM, Vietnam, THA, Thailand, MAL, Malaysia, CHI, People’s Republic of China; TRK, Turkey; RUS, Russia, JPN, Japan. Cambodian subtype H5N1 viruses that are grouped in closely related phylogenetic sublineages and are denoted by sublineage number (arbitrarily numbered I–VII for purposes of molecular epidemiology) and these sublineages are also denoted in the map (Figure 2) to show where these viruses have found and the likely virus introduction routes. It is to be emphasized that all these viruses are within the World Health Organization (WHO) clade 1, and these sublineage numbers are not to be confused with the WHO clade/subclade nomenclature.

Figure 2

Map of Cambodia showing the locations of influenza A (H5N1) outbreaks in poultry (circles) and human cases (stars) detected since 2005. Arrows are proposed to illustrate the hypothetical paths of introduction of H5N1 virus sublineages (see Figure 1) in Cambodia from its neighboring countries. A sublineage number adjacent to the arrow implies that the respective sublineage viruses are found at the start and the end of the arrow with the years of the detection noted (Appendix Table). Molecular characteristics of hemagluttinin (HA) sequence and hemagglutination inhibition (HI) tests of Cambodian viruses and some reference subtype H5N1 strains. A) H5 aa numbering. RBS, receptor binding site; DEL, deletion. B) BHG, bar-headed goose; VNM, Vietnam; CAM, Cambodia; INDO, Indonesia. Numbers I–VII refer to sublineages; within sublineage VII, PV06: group of viruses represented by the strain A/Duck/Cambodia/D1PV/2006; KC06: group of viruses represented by the strain A/Duck/Cambodia/D1KC1672006; CAM07: group of viruses represented by the strain A/Cambodia/R0405050/07; VNM06–07: group of viruses isolated in South Vietnam in 2006 and 2007. C) Antigenic characterization was performed by using the HI assay with ferret antisera raised to World Health Organization reference subtype H5N1 viruses. Numbers are the results of the differences between the log₂(HI titer/10) of the reference virus and the virus tested. D) For HI tests, the virus A/BHG/Qinghai Lake/1A/2005 was tested against this homologous serum, while ferret serum against A/Turkey/15/2005 was tested against the reassortant virus NIBRG-23 derived from A/Turkey/15/2005. A/BHG/Qinghai Lake/1A/2005 and A/Turkey/15/05 are both clade 2.2 viruses.