High pathogenicity avian influenza (HPAI) H5N1 virus is a subtype of the influenza A virus that primarily infects birds. A novel genotype of HPAI H5N1 arose in China in 1996 and has since spread to other countries in Asia, Europe, and Africa. HPAI H5N1 viruses are highly transmissible and lethal in avian species, especially in gallinaceous domestic poultry. HPAI H5N1 virus can also infect humans occasionally. To date, there have been 868 human cases of H5N1 infections globally, 457 of which died from infection with a mortality rate of approximately 53%. Human infection occurs via proximity contact with infected birds, virus-contaminated surfaces, or respiratory secretions. Family clustering of the HPAI H5N1 virus has been reported and raised concerns about human-to-human transmission and thus possible pandemic potential.

On 23 February 2023, a lethal H5N1 human infection case was reported in Prey Veng province, southern Cambodia. The victim was identified as an 11-year-old girl. The father of the index case was also infected and remained asymptomatic. All 11 close contacts tested negative for H5N1. Sequence analysis shows the H5N1 virus belongs to clade 2.3.2.1c, which has been circulating in poultry in southeast Asia since 2014. These are the first two human cases of HPAI H5N1 infections reported from Cambodia since 2014. From 2003 to 25 February 2023, there have been a total of 114 human infections of the H5N1 virus reported in Cambodia, and 75 cases were found to be lethal.The haemagglutinin (HA) of the avian influenza virus (AIV) plays a key role in the interspecies transmission. The shift from avian to human receptor binding preferences, along with increased acid and thermal stability of HA, has been closely associated with human pandemic potential of avian influenza viruses. This association is further supported by the transmission study of H5N1 AIVs using the ferret model. To investigate the zoonotic risk of the human H5N1 isolate, we generated the recombinant viruses via reverse genetics (RG) containing HA and neuraminidase (NA) from the clade 2.3.2.1c HPAI H5N1 viruses and the six internal segments from laboratory adapted A/Puerto Rico/8/34 (H1N1) (PR8) virus. We investigated the risk of the recent H5N1 AIV (A/Cambodia/NPH230032/2023, referred to as KHM/23) to human and animal health by assessing the virus receptor binding, pH of fusion, HA thermal stability, and the antigenicity change with reference to earlier clade 2.3.2.1c HPAI H5N1 virus (A/duck/Vietnam/OIE-2202/2012, referred to as VNM/12).