The genomic landscape of swine influenza A viruses in Southeast Asia.

Swine are a primary source for the emergence of pandemic influenza A viruses. The intensification of swine production, along with global trade, has amplified the transmission and zoonotic risk of swine influenza A virus (swIAV). Effective surveillance is essential to uncover emerging virus strains; however gaps remain in our understanding of the swIAV genomic landscape in Southeast Asia. More than 4,000 nasal swabs were collected from pigs in Cambodia, yielding 72 IAV-positive samples by RT-qPCR and 45 genomic sequences. We unmasked the cocirculation of multiple lineages of genetically diverse swIAV of pandemic concern. Genomic analyses revealed a novel European avian-like H1N2 swIAV reassortant variant with North American triple reassortant internal genes, that emerged approximately seven years before its first detection in pigs in 2021. Using phylogeographic reconstruction, we identified south central China as the dominant source of swine viruses disseminated to other regions in China and Southeast Asia. We also identified nine distinct swIAV lineages in Cambodia, which diverged from their closest ancestors between two and 15 B.P., indicating significant undetected diversity in the region, including reverse zoonoses of human H1N1/2009 pandemic and H3N2 viruses. A similar period of cryptic circulation of swIAVs occurred in the decades before the H1N1/2009 pandemic. The hidden diversity of swIAV observed here further emphasizes the complex underlying evolutionary processes present in this region, reinforcing the importance of genomic surveillance at the human-swine interface for early warning of disease emergence to avoid future pandemics.

Co-circulation of Influenza A H5, H7, and H9 Viruses and Co-infected Poultry in Live Bird Markets, Cambodia.

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.

Isolation and full-genome sequences of Japanese encephalitis virus genotype I strains from Cambodian human patients, mosquitoes and pigs.

Japanese encephalitis remains the most important cause of viral encephalitis in humans in several southeast Asian countries, including Cambodia, causing at least 65 000 cases of encephalitis per year. This vector-borne viral zoonosis - caused by Japanese encephalitis virus (JEV) - is considered to be a rural disease and is transmitted by mosquitoes, with birds and pigs being the natural reservoirs, while humans are accidental hosts. In this study we report the first two JEV isolations in Cambodia from human encephalitis cases from two studies on the aetiology of central nervous system disease, conducted at the two major paediatric hospitals in the country. We also report JEV isolation from Culextritaeniorhynchus mosquitoes and from pig samples collected in two farms, located in peri-urban and rural areas. Out of 11 reverse-transcription polymerase chain reaction-positive original samples, we generated full-genome sequences from 5 JEV isolates. Five additional partial sequences of the JEV NS3 gene from viruses detected in five pigs and one complete coding sequence of the envelope gene of a strain identified in a pig were generated. Phylogenetic analyses revealed that JEV detected in Cambodia belonged to genotype I and clustered in two clades: genotype I-a, mainly comprising strains from Thailand, and genotype I-b, comprising strains from Vietnam that dispersed northwards to China. Finally, in this study, we provide proof that the sequenced JEV strains circulate between pigs, Culex tritaeniorhynchus and humans in the Phnom Penh vicinity.

Environmental contamination and risk factors for transmission of highly pathogenic avian influenza A(H5N1) to humans, Cambodia, 2006-2010.

BACKGROUND: Highly pathogenic avian influenza A (H5N1) virus has been of public health concern since 2003. Probable risk factors for A(H5N1) transmission to human have been demonstrated in several studies or epidemiological reports. However, transmission patterns may differ according to demographic characteristics of the population and local practices. This article aggregates these data from three studies with data collected in the previous surveys in 2006 and 2007 to further examine the risks factors associated with presence of anti-A(H5) antibodies among villagers residing within outbreak areas. METHODS: We aggregated 5-year data (2006-2010) from serology survey and matched case-control studies in Cambodia to further examine the risks factors associated with A(H5N1) infection among villagers in the outbreak areas. RESULTS: Serotesting among villagers detected 35 (1.5 % [0-2.6]) positive cases suggesting recent exposure to A(H5N1) virus. Practices associated with A(H5N1) infection among all ages were: having poultry cage or nesting area under or adjacent to the house (OR: 6.7 [1.6-28.3]; p = 0.010) and transporting poultry to market (OR: 17.6 [1.6-193.7]; p = 0.019). Practices found as risk factors for the infection among age under 20 years were swimming/bathing in ponds also accessed by domestic poultry (OR: 4.6 [1.1-19.1]; p = 0.038). Association with consuming wild birds reached borderline significance (p = 0.066). CONCLUSION: Our results suggest that swimming/bathing in contaminated pond water and close contact with poultry may present a risk of A(H5N1) transmission to human.

Identification of molecular markers associated with alteration of receptor-binding specificity in a novel genotype of highly pathogenic avian influenza A(H5N1) viruses detected in Cambodia in 2013.

Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with the alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to the absence of the mutations in poultry/environmental samples, suggested that the mutations occurred during human infection and did not transmit further.

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.

Detection and phylogenetic analysis of contemporary H14N2 Avian influenza A virus in domestic ducks in Southeast Asia (Cambodia).

Avian influenza virus (AIV) in Asia is a complex system with numerous subtypes and a highly porous wild birds-poultry interface. Certain AIV subtypes, such as H14, are underrepresented in current surveillance efforts, leaving gaps in our understanding of their ecology and evolution. The detection of rare subtype H14 in domestic ducks in Southeast Asia comprises a geographic region and domestic bird population previously unassociated with this subtype. These H14 viruses have a complex evolutionary history involving gene reassortment events. They share sequence similarity to AIVs endemic in Cambodian ducks, and Eurasian low pathogenicity and high pathogenicity H5Nx AIVs. The detection of these H14 viruses in Southeast Asian domestic poultry further advances our knowledge of the ecology and evolution of this subtype and reinforces the need for continued, longitudinal, active surveillance in domestic and wild birds. Additionally, in vivo and in vitro risk assessment should encompass rare AIV subtypes, as they have the potential to establish in poultry systems.

Influenza A(H5N1) virus surveillance at live poultry markets, Cambodia, 2011.

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.

Molecular epidemiological investigation of velogenic Newcastle disease viruses from village chickens in Cambodia.

Three isolates of Newcastle disease virus (NDV) were isolated from tracheal samples of dead village chickens in two provinces (Phnom Penh and Kampong Cham) in Cambodia during 2011-2012. All of these Cambodian NDV isolates were categorized as velogenic pathotype, based on in vivo pathogenicity tests and F cleavage site motif sequence ((112)RRRKRF(117)). The phylogenetic analysis and the evolutionary distances based on the sequences of the F gene revealed that all the three field isolates of NDV from Cambodia form a distinct cluster (VIIh) together with three Indonesian strains and were assigned to the genotype VII within the class II. Further phylogenetic analysis based on the hyper-variable region of the F gene revealed that some of NDV strains from Malaysia since the mid-2000s were also classified into the VIIh virus. This indicates that the VIIh NDVs are spreading through Southeast Asia. The present investigation, therefore, emphasizes the importance of further surveillance of NDV in neighboring countries as well as throughout Southeast Asia to contain further spreading of these VIIh viruses.

Phylogenetic analysis and genetic characterization of chicken anemia virus isolates from Cambodia.

Three chicken anemia viruses (CAV) were detected by PCR during screening of field samples from village chickens collected in Cambodia in 2011/2012. Nearly full-length VP1 viral structural protein genes (nt 1-1,293) from the 3 CAV were sequenced and characterized. Phylogenetic analysis revealed that all 3 of the Cambodian CAV were clustered with CAV strains belonging to genotype II and were most closely related to CAV strains from Guangdong province, China. On the amino acid level, major substitutions were observed at 12 residues in the VP1 protein (positions 22, 75, 97, 125, 139, 144, 254, 287, 290, 370, 376, and 413) when compared with published reference CAV strains. In motifs associated with virulence, all Cambodian CAV had virulence-associated motifs composed of 75I, 89T, 125I, 139Q, 141Q, 144Q, and 394Q, which are commonly found in highly virulent genotype II viruses and some genotype III viruses. This is the first report of CAV isolated from village chickens in Southeast Asia as well as Cambodia.

Case report: Lumpy skin disease in an endangered wild banteng (Bos javanicus) and initiation of a vaccination campaign in domestic livestock in Cambodia.

We describe a case of lumpy skin disease in an endangered banteng in Cambodia and the subsequent initiation of a vaccination campaign in domestic cattle to protect wild bovids from disease transmission at the wildlife-livestock interface. Lumpy skin disease virus (LSDV) was first detected in domestic cattle in Cambodia in June of 2021 and rapidly spread throughout the country. In September 2021, a banteng was seen in Phnom Tnout Phnom Pok wildlife sanctuary with signs of lumpy skin disease. Scab samples were collected and tested positive for LSDV. Monitoring using line transect surveys and camera traps in protected areas with critical banteng and gaur populations was initiated from December 2021-October 2022. A collaborative multisector vaccination campaign to vaccinate domestic livestock in and around priority protected areas with banteng and gaur was launched July 2022 and a total of 20,089 domestic cattle and water buffalo were vaccinated with LumpyvaxTM. No signs of LSDV in banteng or gaur in Cambodia have been observed since this initial case. This report documents the first case of lumpy skin disease in wildlife in Cambodia and proposes a potential intervention to mitigate the challenge of pathogen transmission at the domestic-wildlife interface. While vaccination can support local livestock-based economies and promote biodiversity conservation, it is only a component of an integrated solution and One Health approach to protect endangered species from threats at the wildlife-livestock interface.

Biosecurity measures for backyard poultry in developing countries: a systematic review.

BACKGROUND: Poultry represents an important sector in animal production, with backyard flocks representing a huge majority, especially in the developing countries. In these countries, villagers raise poultry to meet household food demands and as additional sources of incomes. Backyard production methods imply low biosecurity measures and high risk of infectious diseases, such as Newcastle disease or zoonosis such as Highly Pathogenic Avian Influenza (HPAI).We reviewed literature on biosecurity practices for prevention of infectious diseases, and published recommendations for backyard poultry and assessed evidence of their impact and feasibility, particularly in developing countries. Documents were sourced from the Food and Agriculture Organization (FAO) website, and from Pubmed and Google databases. RESULTS: A total of 62 peer-reviewed and non-referred documents were found, most of which were published recently (after 2004) and focused on HPAI/H5N1-related biosecurity measures (64%). Recommendations addressed measures for flock management, feed and water management, poultry trade and stock change, poultry health management and the risk to humans. Only one general guideline was found for backyard poultry-related biosecurity; the other documents were drawn up for specific developing settings and only engaged their authors (e.g. consultants). These national guidelines written by consultants generated recommendations regarding measures derived from the highest standards of commercial poultry production. Although biosecurity principles of isolation and containment are described in most documents, only a few documents were found on the impact of measures in family poultry settings and none gave any evidence of their feasibility and effectiveness for backyard poultry. CONCLUSIONS: Given the persistent threat posed by HPAI/H5N1 to humans in developing countries, our findings highlight the importance of encouraging applied research toward identifying sustained and adapted biosecurity measures for smallholder poultry flocks in low-income countries.

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.

Highly pathogenic avian influenza virus (H5N1) outbreak in captive wild birds and cats, Cambodia.

From December 2003 through January 2004, the Phnom Tamao Wildlife Rescue Centre, Cambodia, was affected by the highly pathogenic influenza virus (H5N1). Birds from 26 species died. Influenza virus subtype H5N1 was detected in 6 of 7 species tested. Cats from 5 of 7 species were probably infected; none died.

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.

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/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.

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.

Comparison of the dynamics of Japanese encephalitis virus circulation in sentinel pigs between a rural and a peri-urban setting in Cambodia.

Japanese encephalitis is mainly considered a rural disease, but there is growing evidence of a peri-urban and urban transmission in several countries, including Cambodia. We, therefore, compared the epidemiologic dynamic of Japanese encephalitis between a rural and a peri-urban setting in Cambodia. We monitored two cohorts of 15 pigs and determined the force of infection-rate at which seronegative pigs become positive-in two study farms located in a peri-urban and rural area, respectively. We also studied the mosquito abundance and diversity in proximity of the pigs, as well as the host densities in both areas. All the pigs seroconverted before the age of 6 months. The force of infection was 0.061 per day (95% confidence interval = 0.034-0.098) in the peri-urban cohort and 0.069 per day (95% confidence interval = 0.047-0.099) in the rural cohort. Several differences in the epidemiologic dynamic of Japanese encephalitis between both study sites were highlighted. The later virus amplification in the rural cohort may be linked to the later waning of maternal antibodies, but also to the higher pig density in direct proximity of the studied pigs, which could have led to a dilution of mosquito bites at the farm level. The force of infection was almost identical in both the peri-urban and the rural farms studied, which shifts the classic epidemiologic cycle of the virus. This study is a first step in improving our understanding of Japanese encephalitis virus ecology in different environments with distinct landscapes, human and animal densities.

Influenza A(H5N1) viruses with A(H9N2) single gene (matrix or PB1) reassortment isolated from Cambodian live bird markets.

Live bird market surveillance for avian influenza viruses in Cambodia in 2015 has led to the detection of two 7:1 reassortant influenza A(H5N1) clade 2.3.2.1c viruses. These reassortant strains, designated A/duck/Cambodia/Z564W35M1/2015 and A/chicken/Cambodia/Z850W49M1/2015, both contained a single gene (PB1 and matrix gene, respectively) from concurrently circulating A(H9N2) influenza viruses. All other viral genes from both isolates clustered with A(H5N1) clade 2.3.2.1 viruses. Continued and prolonged co-circulation of influenza A(H5N1) and A(H9N2) viruses in Cambodian live bird markets may present a risk for the emergence of novel influenza reassortant viruses with negative agricultural and/or public health implications.

Effects of anthelmintic treatment and feed supplementation on parasite infections and morbidity parameters in Cambodian cattle.

Helminth infections are the cause of morbidity in Cambodian cattle but other factors such as nutritional deficiencies and concurrent diseases may enhance the effects of parasites. The present study aimed to investigate the impact of anthelmintic treatment, feed supplementation, or both on gastrointestinal strongyle (GIS) and trematode infections as well as on morbidity parameters in Cambodian village cattle. At the beginning of the dry season, cattle populations in six villages were randomly assigned to a group: (A) receiving anthelmintic treatment (ivermectin+clorsulon) at week 0; (P) feed pellet supplementation during week 0-13 or both (AP). On five visits (week 0-29), faecal and blood samples were obtained for parasitological examination and haematocrit determination, respectively. Body condition (BCS), hind quarter fouling (HQFS), diarrhoea (DS), and conjunctiva colour (FAMACHA©) were scored and heart girth circumference was determined. To investigate the impact of treatment over time (week 0-29), a mixed model was used with treatment, time, and their interaction as fixed effects, and animal and village as random factors. At baseline, the proportion of GIS positive animals was high (67.9%), whereas trematode infections were low (Paramphistomum: 8.8%; Fasciola: 2.6%). Very thin to emaciated cattle (BCS 1-2) were more prevalent (11.4%) and FAMACHA© scores of ≤3 or below (65.8%) less prevalent than in an earlier study in the region. A Time ⨯ Treatment interaction was present for faecal egg counts (FEC) of GIS, GIS prevalence (both p<0.0001), PCV (p=0.0034), DS (p=0.0086) and HQFS (p=0.0241). For GIS FEC, treatment groups differed at a specific time point, with levels of treatment group P being higher than in A at week 6 (p=0.0054). For Paramphistomum prevalence as well as FAMACHA© scoring, heart girth and BCS, the interaction between treatment and time was not significant, yet, time in itself had a significant impact on all (p<0.0001). The beneficial effects of protein supplementation were unclear from the current study.