Longitudinal surveillance of 2 live bird markets in Cambodia revealed year-round, high co-circulation of H5, H7, and H9 influenza viruses. We detected influenza A viruses in 51.3% of ducks and 39.6% ...of chickens, and co-infections, mainly by H5 and H9 viruses, in 0.8% of ducks and 4.5% of chickens.
In Cambodia, influenza A(H5N1) virus surveillance at live poultry markets (LPMs) relies on virus isolation from poultry specimens; however, virus is rarely detected by this method. We tested 502 ...environmental LPM samples: 90 were positive by PCR, 10 by virus isolation. Virus circulation could be better monitored by environmental sampling of LPMs.
Live bird markets (LBMs) have been identified as key factors in the spread, persistence and evolution of avian influenza viruses (AIVs). In addition, these settings have been associated with human ...infections with AIVs of pandemic concern. Exposure to aerosolised AIVs by workers in a Cambodian LBM was assessed using aerosol impact samplers. LBM vendors were asked to wear an air sampler for 30 min per day for 1 week while continuing their usual activities in the LBM during a period of high AIV circulation (February) and a period of low circulation (May). During the period of high circulation, AIV RNA was detected from 100% of the air samplers using molecular methods and viable AIV (A/H5N1 and/or A/H9N2) was isolated from 50% of air samplers following inoculation into embryonated chicken eggs. In contrast, AIV was not detected by molecular methods or successfully isolated during the period of low circulation. This study demonstrates the increased risk of aerosol exposure of LBM workers to AIVs during periods of high circulation and highlights the need for interventions during these high‐risk periods. Novel approaches, such as environmental sampling, should be further explored at key high‐risk interfaces as a potentially cost‐effective alternative for monitoring pandemic threats.
Avian influenza viruses (AIVs) periodically cross species barriers and infect humans. The likelihood that an AIV will evolve mammalian transmissibility depends on acquiring and selecting mutations ...during spillover, but data from natural infection is limited. We analyze deep sequencing data from infected humans and domestic ducks in Cambodia to examine how H5N1 viruses evolve during spillover. Overall, viral populations in both species are predominated by low-frequency (<10%) variation shaped by purifying selection and genetic drift, and half of the variants detected within-host are never detected on the H5N1 virus phylogeny. However, we do detect a subset of mutations linked to human receptor binding and replication (PB2 E627K, HA A150V, and HA Q238L) that arose in multiple, independent humans. PB2 E627K and HA A150V were also enriched along phylogenetic branches leading to human infections, suggesting that they are likely human-adaptive. Our data show that H5N1 viruses generate putative human-adapting mutations during natural spillover infection, many of which are detected at >5% frequency within-host. However, short infection times, genetic drift, and purifying selection likely restrict their ability to evolve extensively during a single infection. Applying evolutionary methods to sequence data, we reveal a detailed view of H5N1 virus adaptive potential, and develop a foundation for studying host-adaptation in other zoonotic viruses.
Low pathogenic A(H9N2) subtype avian influenza viruses (AIVs) were originally detected in Cambodian poultry in 2013, and now circulate endemically. We sequenced and characterised 64 A(H9N2) AIVs ...detected in Cambodian poultry (chickens and ducks) from January 2015 to May 2016. All A(H9) viruses collected in 2015 and 2016 belonged to a new BJ/94-like h9-4.2.5 sub-lineage that emerged in the region during or after 2013, and was distinct to previously detected Cambodian viruses. Overall, there was a reduction of genetic diversity of H9N2 since 2013, however two genotypes were detected in circulation, P and V, with extensive reassortment between the viruses. Phylogenetic analysis showed a close relationship between A(H9N2) AIVs detected in Cambodian and Vietnamese poultry, highlighting cross-border trade/movement of live, domestic poultry between the countries. Wild birds may also play a role in A(H9N2) transmission in the region. Some genes of the Cambodian isolates frequently clustered with zoonotic A(H7N9), A(H9N2) and A(H10N8) viruses, suggesting a common ecology. Molecular analysis showed 100% of viruses contained the hemagglutinin (HA) Q226L substitution, which favours mammalian receptor type binding. All viruses were susceptible to the neuraminidase inhibitor antivirals; however, 41% contained the matrix (M2) S31N substitution associated with resistance to adamantanes. Overall, Cambodian A(H9N2) viruses possessed factors known to increase zoonotic potential, and therefore their evolution should be continually monitored.
In Cambodia, highly pathogenic avian influenza A(H5N1) subtype viruses circulate endemically causing poultry outbreaks and zoonotic human cases. To investigate the genomic diversity and development ...of endemicity of the predominantly circulating clade 2.3.2.1c A(H5N1) viruses, we characterised 68 AIVs detected in poultry, the environment and from a single human A(H5N1) case from January 2014 to December 2016. Full genomes were generated for 42 A(H5N1) viruses. Phylogenetic analysis shows that five clade 2.3.2.1c genotypes, designated KH1 to KH5, were circulating in Cambodia during this period. The genotypes arose through multiple reassortment events with the neuraminidase (NA) and internal genes belonging to H5N1 clade 2.3.2.1a, clade 2.3.2.1b or A(H9N2) lineages. Phylogenies suggest that the Cambodian AIVs were derived from viruses circulating between Cambodian and Vietnamese poultry. Molecular analyses show that these viruses contained the hemagglutinin (HA) gene substitutions D94N, S133A, S155N, T156A, T188I and K189R known to increase binding to the human-type α2,6-linked sialic acid receptors. Two A(H5N1) viruses displayed the M2 gene S31N or A30T substitutions indicative of adamantane resistance, however, susceptibility testing towards neuraminidase inhibitors (oseltamivir, zanamivir, lananmivir and peramivir) of a subset of thirty clade 2.3.2.1c viruses showed susceptibility to all four drugs. This study shows that A(H5N1) viruses continue to reassort with other A(H5N1) and A(H9N2) viruses that are endemic in the region, highlighting the risk of introduction and emergence of novel A(H5N1) genotypes in Cambodia.
In January 2017, an estimated 3,700 (93%) of 4,000 Khaki Campbell ducks (Anas platyrhynchos domesticus) died in Kampong Thom Province, Cambodia. We detected low pathogenicity avian influenza A(H7N3) ...virus and anatid herpesvirus 1 (duck plague) in the affected flock; however, the exact cause of the mortality event remains unclear.
Very little is known regarding the persistence of Highly Pathogenic Avian Influenza (HPAI) H5N1 viruses in aquatic environments in tropical countries, although environmental materials have been ...suggested to play a role as reservoirs and sources of transmission for H5N1 viruses.
The survival of HPAI H5N1 viruses in experimental aquatic biotopes (water, mud, aquatic flora and fauna) relevant to field conditions in Cambodia was investigated. Artificial aquatic biotopes, including simple ones containing only mud and water, and complex biotopes involving the presence of aquatic flora and fauna, were set up. They were experimentally contaminated with H5N1 virus. The persistence of HPAI H5N1 virus (local avian and human isolates) was determined by virus isolation in embryonated chicken eggs and by real-time reverse-polymerase chain reaction. Persistence of infectious virus did not exceed 4 days, and was only identified in rain water. No infectious virus particles were detected in pond and lake water or mud even when high inoculum doses were used. However, viral RNA persisted up to 20 days in rain water and 7 days in pond or lake water. Viral RNA was also detected in mud samples, up to 14 days post-contamination in several cases. Infectious virus and viral RNA was detected in few cases in the aquatic fauna and flora, especially in bivalves and labyrinth fish, although these organisms seemed to be mostly passive carriers of the virus rather than host allowing virus replication.
Although several factors for the survival and persistence of HPAI viruses in the environment are still to be elucidated, and are particularly hard to control in laboratory conditions, our results, along with previous data, support the idea that environmental surveillance is of major relevance for avian influenza control programs.
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