Intense circulation of A/H5N1 and other avian influenza viruses in Cambodian live-bird markets with serological evidence of sub-clinical human infections

Abstract

Surveillance for avian influenza viruses (AIVs) in poultry and environmental samples was conducted in four live-bird markets in Cambodia from January through November 2013. Through real-time RT-PCR testing, AIVs were detected in 45% of 1048 samples collected throughout the year. Detection rates ranged from 32% and 18% in duck and chicken swabs, respectively, to 75% in carcass wash water samples. Influenza A/H5N1 virus was detected in 79% of samples positive for influenza A virus and 35% of all samples collected. Sequence analysis of full-length haemagglutinin (HA) and neuraminidase (NA) genes from A/H5N1 viruses, and full-genome analysis of six representative isolates, revealed that the clade 1.1.2 reassortant virus associated with Cambodian human cases during 2013 was the only A/H5N1 virus detected during the year. However, multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of HA and NA genes revealed co-circulation of at least nine low pathogenic AIVs from HA1, HA2, HA3, HA4, HA6, HA7, HA9, HA10 and HA11 subtypes. Four repeated serological surveys were conducted throughout the year in a cohort of 125 poultry workers. Serological testing found an overall prevalence of 4.5% and 1.8% for antibodies to A/H5N1 and A/H9N2, respectively. Seroconversion rates of 3.7 and 0.9 cases per 1000 person-months participation were detected for A/H5N1 and A/H9N2, respectively. Peak AIV circulation was associated with the Lunar New Year festival. Knowledge of periods of increased circulation of avian influenza in markets should inform intervention measures such as market cleaning and closures to reduce risk of human infections and emergence of novel AIVs.

Figure 1

Map showing communes with reported confirmed human A/H5N1 cases during 2006–2014, population density (indicated by the number of villages) and live-bird markets investigated in 2013.

Figure 2

Live-bird markets in Cambodia typically have poor biosecurity, with multiple animal species slaughtered onsite. Animal and environmental sampling in these markets is crucial to monitor virus circulation and the emergence/introduction of new viruses.

Figure 3

Proportion of samples positive for Influenza A virus (M-gene) for each sampling mission at four live-bird markets in Cambodia, 2013. The major Cambodian festivals are during week 6 (Lunar New Year festival), week 15 (Khmer New Year festival) and week 40 (Pchum Ben festival), and are indicated by vertical blue bars.

Figure 4

Neighbour-joining phylogenetic tree of the HA and NA genes of highly pathogenic avian influenza A/H5N1 viruses detected during live-bird market surveillance in Cambodia. Viruses collected during the present market study are denoted by a black circle. Viruses for which the full genome is available are denoted in bold. Mid-point rooted phylogenetic trees were constructed in MEGA5. Bootstraps >70 generated from 1000 replicates are shown at branch nodes. The scale bar represents the number of nucleotide substitutions per site.

Figure 5

Heat map of the avian influenza virus positivity rate in four Cambodian live-poultry markets, 2013.