International Research in Cambodia: Tropical Medicine and Vector Research

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

The NIAID Division of Intramural Research International Centers for Excellence in Research (ICER) in Cambodia is a DIR program focused on the studies of infectious diseases, immunology, and vector biology in Cambodia. The scope of studies at ICER Cambodia includes vector-borne diseases, antimicrobial resistance, emerging infections, and zoonotic diseases. The overarching goal of the ICER Cambodia program is to enable development of new or improved diagnostics and therapeutic countermeasures by studying these diseases where they are most active, to protect the health of Americans and contain infectious threats before they breach US borders. A DIVERSE PATHOGEN LANDSCAPE IS RESPONSIBLE FOR ACUTE FEBRILE ILLNESS IN CAMBODIA. In 2019, we implemented an in-country pathogen short-read sequencing pipeline for evaluation of clinical samples from febrile individuals aged 2 months to 65 years of age in Cambodia (NCT04034264). As of 2025, mNGS analysis of over 3000 individuals identified vector-borne pathogens as the largest clinical category responsible for febrile disease (Bohl PNAS 2022). Dengue virus (DENV) was the most common, in part due to a historic outbreak in 2019 that was potentially driven by introduction of a new genotype (Brook PNAS 2024). Metagenomic sequencing of sera samples enabled identification of chikungunya virus that presaged a larger national outbreak in 2020, leading to vector control interventions and the addition of chikungunya PCR to the national surveillance system. Interestingly, underappreciated vector-borne and zoonotic pathogens such as Plasmodium knowlesi, Rickettsia spp., scrub typhus, leptospirosis, and co-infecting HIV were also detected (Yek AJTMH 2022). Among febrile persons with positive clinical cultures, untargeted sequencing approaches identified a diverse range of bacterial pathogens, among which the three most common were Escherichia coli, Acinetobacter baumannii, and Burkholderia pseudomallei. Partial and whole genome data generated through this approach enabled pathogen characterization including identification of genetic markers of antimicrobial resistance, recognition of disease outbreaks, and descriptions of pathogen evolution and phylogeography (Yek JID 2024). We demonstrated that untargeted sequencing approaches can be used to identify and monitor the breadth of pathogens leading to acute febrile illness, and in turn inform development of diagnostic algorithms and tailored solutions. EVOLUTIONARY BOTTLENECKS IN SEASONAL INFLUENZA CASES SUGGEST EXTERNAL INTRODUCTION EVENTS IN 2022. Nasopharyngeal sampling of acute febrile individuals (NCT04034264) from 2020-2023 captured a variety of respiratory viruses in Cambodia during the height of the COVID-19 pandemic. As pandemic-related societal mobility restrictions were lifted, there was a brisk resurgence of respiratory viruses with cases increasing by over 4-fold. Both SARS-CoV-2 and non-SARS-CoV-2 cases rose, the former dominated by the highly transmissible Omicron variant and the latter initially by diverse Enteroviruses. Our data suggest that Cambodia may have experienced a ‘tripledemic’ of SARS-CoV-2, influenza, and RSV in mid- to late- 2022. We were able to identify monophyletic re-emergences of Influenza A and B (A/H3N2/3C.2a1b.2a.2a and B/Victoria/1A.3a.2, respectively) and found that these Cambodian diverged from pre-pandemic strains in Cambodia and lagged homologous seasons in other continents. Taken together, these findings are consistent with external introduction events rather than cryptic circulation (findings presented at the 2024 ASTMH Annual Meeting). CAMBODIAN INVASIVE BURKHOLDERIA PSEUDOMALLEI SEQUENCE TYPES ARE BROADLY DISPERSED AND DEMONSTRATE UNIQUE GENOMIC FEATURES. Between 2021-2023, we collected and sequenced 253 clinical B. pseudomallei isolates from patients admitted to six Cambodian hospitals. We compared geographic distributions of assembled genome sequence types and clades, revealing signals of clustering in the Mekong Delta south of Phnom Penh and around the Tonle Sap to the north. We described unique genomic features within the isolate cohort, including mutually exclusive presence of genes associated with antimicrobial resistance, and identification of a chromosomal metabolic genomic island among isolates mapping to one clade. Finally, we supplemented these analyses with 3,234 global isolate genomes from public repositories, which revealed intermixing between Cambodian and other Southeast Asian isolates but clear distinction from Australian strains (findings presented at the 2024 ASTMH Annual Meeting).  NON-HUMAN PRIMATES PLAY A LIMITED ROLE IN SUSTAINING AND AMPLIFYING URBAN ARBOVIRAL CYCLES IN CAMBODIA. Between 2019 and 2022, we collaborated with investigators at Mahidol University in Bangkok, Thailand to conduct cross-sectional serosurveys of 649 human adults and 398 long-tailed macaques living in proximity across four sites in Cambodia and Thailand (NCT04434846). We sought to use these data to explore potential impacts of arboviral circulation within sylvatic reservoirs on human arboviral disease dynamics. We performed plaque reduction neutralization tests in human and NHP sera and found overall higher seropositivity rates across arboviruses among human populations compared to NHP (38.5-74.4% versus 0-8.0% respectively, using PRNT50 cutoffs). Virus seroprevalence differed between Thai and Cambodian cohorts and age was the only significant predictor of human seropositivity in multivariate analyses. These findings suggest a limited role of NHPs in sustaining and amplifying urban arboviral cycles; rather, low-level NHP seropositivity may suggest virus spillback from human populations (Mongkol JID 2025). PRODUCTION OF IFN-ALPHA BY HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS IS SUPPRESSED BY AEDES AEGYPTI SALIVA. When arboviruses like dengue infect human cells, they trigger Interferon type I responses, particularly IFN-alpha (IFN-α). IFN-α can act on both virus-infected and neighboring cells by activating their cellular defense mechanisms, thereby inhibiting viral expansion. However, during natural transmission of arboviruses, the virus is co-injected with mosquito saliva, which contains over 100 pharmacologically active compounds. Our research has revealed that Aedes aegypti saliva can modulate IFN-α production, potentially facilitating viral growth and exacerbating the infection (findings presented at the 2024 ASTMH Annual Meeting). We are currently investigating which specific cell types are most affected by this suppression and identifying the salivary proteins responsible for this effect. ANTIBODY DISCOVERY IN EMERGING INFECTIONS. Since February 2023, there have been 28 PCR-confirmed human cases of Highly Pathogenic Avian Influenza (HPAI) resulting from infection with Influenza A/ H5N1. Of these, seven cases have been enrolled (NCT04034264) with a goal to isolate neutralizing monoclonal antibodies. To date, a mAb targeting NA, termed Cam23-2B05, was isolated from a Cambodian survivor of clade 2.3.2.1e H5N1 infection. Cam23-2B05 displays broad binding and NA inhibitory activity across avian H5N1 and human H1N1 viruses. Cryo-electron microscopy structure reveals that Cam23-2B05 recognizes a conserved quaternary epitope spanning two adjacent NA protomers on the lateral surface of the globular head, distal from the catalytic site. The Cam23-2B05 epitope partly overlaps with those of previously identified mAbs, such as murine CD6, human 3H03 and 2H08, suggesting that this epitope is an antigenic supersite. Furthermore, Cam23-2B05 inhibits NA activity through steric hindrance and confers prophylactic protection against a lethal H1N1 (A/Puerto Rico/8/1934) challenge in mice (findings presented at the 2025 Keystone Conference in Vaccinology). In two mixed adult and pediatric cohorts sampled adventitiously prior to the SARS-CoV-2 pandemic (total N=821), we explored humoral immunity to SARS-CoV-2 and found 7-14% seroprevalence that could not be explained by immunity against seasonal betacoronaviruses (Manning EID 2022) or Plasmodium falciparum (Fathi ADV SCI 2025). We collaborated with investigators at the National University of Singapore to screen samples for neutralizing activity against a panel of human and animal sarbecoviruses, merbecoviruses, and henipaviruses, as part of a multi-site study to validate a high-throughput surrogate virus neutralization test. Samples collected in Southeast Asian countries exhibited higher seropositive rates for SARS-like viruses than those from New Zealand and Tanzania, suggesting more frequent spillover of related viruses in this region potentially due to abundance of animal reservoirs (manuscript under review). NANOINJECTION TO ACHIEVE AEDES AEGYPTI INFECTION WITH NATURALLY ATTENUATED DENV-2 VIRUS. Several dengue challenge human infection models (CHIMs) have been used to assess the safety and efficacy of dengue vaccines and therapies. However, these have used needle inoculation of the virus, bypassing the natural mosquito vector. We propose to develop a “natural CHIM” using dengue infected Aedes aegypti for human challenge. For safety and feasibility, we chose a naturally attenuated DENV-2 delta 30-7169 virus shown to cause mild symptoms (rash) and viremia, but no severe dengue outcomes in humans. An additional benefit of using this attenuated DENV-2 strain is its poorly transmissibility back to mosquitoes by blood feeding. We injected Aedes aegypti intrathoracically with 100 versus 300 DENV-2 delta 30-7169 plaque forming units and evaluated its dissemination using PCR of dissected mosquito tissues. We found that all tissues had detectable virus by PCR at day 3 post-injection. Viral titers amplified over time, plateauing at day 18 post-injection. Salivary glands accumulated significantly higher viral titers versus midguts and Malpighian tubules from day 7 post injection onwards. Finally, we detected virus in mosquito saliva by PCR and confirmed its infectivity by salivary gland titration in a human cell line. In summary, we show that this naturally attenuated DENV-2 strain can infect and disseminate within Aedes aegypti mosquitoes following intrathoracic inoculation, making this a suitable model to develop a “natural CHIM” using dengue infected mosquitoes (findings presented at the 2024 ASTMH Annual Meeting).