Molecular Epidemiology of Clade 1 Influenza A Viruses (H5N1), Southern Indochina Peninsula, 2004–2007

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.

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. Subtype H5N1 infections were observed primarily during the dry season, from January through April. The origin of these epizootics in Cambodia remains unclear. Here we have used maximum likelihood and Bayesian methods to analyze the Cambodian virus genetic sequences, together with those obtained from other H5N1 viruses isolated in neighboring countries since 2004, to understand that patterns of virus transmission.

The Study
We analyzed the sequences of all 8 genomic segments of 33 subtype H5N1 viruses sampled in Cambodia from 2004 through 2007, together with publicly available sequences from 116 isolates from Southeast Asia obtained from GenBank. We included all subtype H5N1 viruses sequences from southern Vietnam. In Cambodia, we se-quenced systematically at least 2 strains from each infected flock and obtained a total of 137 sequences. When sequences obtained from viruses isolated during the same outbreak site were identical, we used only one for the analyses. After alignment, phylogenetic analyses were conducted by using maximum-likelihood and Bayesian methods (1,2).
All H5N1 viruses that were detected in Cambodia from 2004 through 2007 belong exclusively to clade 1, genotype Z ( Figure 1 For all gene segments, the percentage of homology varied from 98% (for the HA segment) to 99.8% (for the M1 coding gene) between sequences of the first and the latest virus for which complete genome sequences were obtained. Mutations S123P and S129A, R to G substitution in the HA cleavage site, and S155N and K189N mutations at antigenic site B (which could explain the antigenic drift measured) are characteristics that seem to have become established in the latest Cambodian isolates (online Appendix Table, available from www.cdc.gov/EID/ content/15/10/1641-appT.htm).

Conclusions
Cambodia is essentially a poultry-importing country. The first poultry deaths were observed in semi-industrial   (6), Cambodian farmers tend to release ducks in the paddy fields, particularly after harvesting periods in which duck flocks could feed on the harvest's leftovers. Seasonality could then be explained by higher duck density (7,8). Viruses in Cambodia very likely represent multiple external introductions rather than becoming established within Cambodia on a continuous basis. This may be related to the fact that the poultry density in Cambodia is lower (average 94 heads/km 2 ) (9) compared with that in neighboring countries such as Thailand and Vietnam (average 640 heads/km 2 ) (10), and one may speculate that a minimum density of poultry is required for maintaining an endemic virus. Lake Tonle Sap (16,000 km 2 surface) and other lakes and wetlands host numerous wild birds, mostly resident or regional migratory birds (M. Gilbert, pers.comm.), which could be involved in the spread of subtype H5N1 virus. Should this mode of transmission play a role, it would probably be limited to local or regional transmission, especially since viruses from other clades (i.e., clade 2.2) that are imported to neighboring countries by migratory birds from the People's Republic of China, have never been detected in Cambodia.
Sublineage VII-specific mutations (i.e., R325G, S123P) in strains from Cambodia and Vietnam suggest that this new sublineage became endemic in the southern Cambodia-Mekong Delta region during 2006. The phenomenon of virus spread through poultry trading, particularly along the roads, accounts for maintenance and extension of virus within a given region and sustains the risks of transmission to humans.  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 (online Technical Appendix