Serological evidence for Japanese encephalitis and West Nile virus infections in domestic birds in Cambodia

Mosquito-borne flaviviruses with an enzootic transmission cycle like Japanese encephalitis virus (JEV) and West Nile virus (WNV) are a major public health concern. The circulation of JEV in Southeast Asia is well-documented, and the important role of pigs as amplification hosts for the virus is long known. The influence of other domestic animals especially poultry that lives in high abundance and close proximity to humans is not intensively analyzed. Another understudied field in Asia is the presence of the closely related WNV. Such analyses are difficult to perform due to the intense antigenic cross-reactivity between these viruses and the lack of suitable standardized serological assays. The main objective of this study was to assess the prevalence of JEV and WNV flaviviruses in domestic birds, detailed in chickens and ducks, in three different Cambodian provinces. We determined the flavivirus seroprevalence using an hemagglutination inhibition assay (HIA). Additionally, we investigated in positive samples the presence of JEV and WNV neutralizing antibodies (nAb) using foci reduction neutralization test (FRNT). We found 29% (180/620) of the investigated birds positive for flavivirus antibodies with an age-depended increase of the seroprevalence (OR = 1.04) and a higher prevalence in ducks compared to chicken (OR = 3.01). Within the flavivirus-positive birds, we found 43% (28/65) with nAb against JEV. We also observed the expected cross-reactivity between JEV and WNV, by identifying 18.5% double-positive birds that had higher titers of nAb than single-positive birds. Additionally, seven domestic birds (10.7%) showed only nAb against WNV and no nAb against JEV. Our study provides evidence for an intense JEV circulation in domestic birds in Cambodia, and the first serological evidence for WNV presence in Southeast Asia since decades. These findings mark the need for a re-definition of areas at risk for JEV and WNV transmission, and the need for further and intensified surveillance of mosquito-transmitted diseases in domestic animals

Prevalence and associated risk factors of Toxocara vitulorum infections in buffalo and cattle calves in three provinces of Central Cambodia

The prevalence and associated risk factors of Toxocara vitulorum infection in buffalo and cattle calves was studied in 3 provinces in central Cambodia. Fecal samples were collected from 517 calves between the age of 1-15 weeks and processed for nematode egg counts by a modified McMaster method. A total of 64 calves were found to excrete T. vitulorum eggs in their feces (12.4%; 95% exact CI: 9.7-15.5). The mean fecal egg count was 2,798 EPG (SD=16,351; range=0-224,400). A multivariable generalized linear mixed model showed higher odds of T. vitulorum infection for buffalo versus cattle, for animals aged 4-8 weeks versus younger and older ones, and for animals with strongyle infection. There was no association with fecal consistency. Farmers should be aware of the potential impact of T. vitulorum, and treat their calves at the age of 2-3 weeks with anthelmintics such as benzimidazoles or pyrantel

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

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

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

Aerosol exposure of live bird market workers to viable influenza A/H5N1 and A/H9N2 viruses, Cambodia

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

The evolution and genetic diversity of avian influenza A(H9N2) viruses in Cambodia, 2015 – 2016

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/94like 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

Diversity of A(H5N1) clade 2.3.2.1c avian Influenza viruses with evidence of reassortment in Cambodia, 2014-2016

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