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iScience: Multiplex Serological Profiling Reveals Diverse Avian and Mammalian Influenza A Virus Exposure in Swine (in Cambodia)
#19,126
While the spillover of HPAI H5N1 into dairy cattle has garnered considerable attention over the past two years, historically, the susceptibility of swine to diverse influenza A viruses has generated the most pandemic concern.
In May of 2023, in Netherlands: Zoonoses Experts Council (DB-Z) Risk Assessment & Warning of Swine As `Mixing Vessels' For Avian Flu, we looked at growing concerns in Europe that avian H5N1 could increase its pandemic threat by spreading (and evolving) in farmed swine.
Only days later, a report out of Italy confirmed an H5N1 spillover event at a `mixed species' farm (poultry & swine), and the subsequent seroconversion of the majority of the pigs tested on that farm (see Study: Seroconversion of a Swine Herd in a Free-Range Rural Multi-Species Farm against HPAI H5N1 2.3.4.4b Clade Virus).
While HPAI detections in swine have been limited, we've seen scattered evidence that H5N1 can infect pigs, albeit often asymptomatically. A few past reports include:
But testing is extremely limited; PCR testing can only pick up current infection (which may only last days or weeks), while antibody testing (HI/MN/NP-ELISA) can often infer past infections, but are labor intensive and can generally only screen for one subtype at a time.
While RT-PCR can only tell you `what is', and serology only `what was', most countries aren't aggressively testing with either method.
All of which brings us to a new report, published in iScience, which provides a proof-of-concept that a multiplex immunoassay panel can detect a wide range of influenza A subtype antibodies in one pass.
While much of this report will be of interest primarily to lab geeks, the testing took place in Cambodia between 2020 and 2022, which has been the site of a renewed outbreak of HPAI H5N1 since 2023 (see Cambodian MOH Announces 4th Human H5N1 Case of 2026).
While this testing pre-dates this recent resurgence, it revealed a complex panoply of influenza A viruses have circulated in Cambodia pigs, including a faint H5 signal (see graphical abstract below).
The Abstract, and a brief excerpt, from the study follow. I'll have a bit more after the break.
Animal origin influenza viruses pose significant pandemic threats, with swine serving as key hosts. Serological surveillance in pigs remains limited in regions with intense human–animal–avian contact. Between March 2020 and July 2022, we collected 4,089 pig serum samples in Cambodia, of which 1,321 (32.5%) were influenza A virus (IAV) seropositive by ELISA.
We developed a multiplex microsphere immunoassay comprising a broad panel of hemagglutinin (HA) and neuraminidase (NA) antigens.
Seroprevalence was highest for pandemic H1N1/pdm09 (35.3–45.7%) and lower for classical swine H1 (16.4–17.7%) and European avian-like swine H1 (∼15%) lineages.
Lineage-specific or shared exposures to multiple lineages were observed, indicating complex infection histories. H3 responses varied by clade, while antibodies to avian H5, H7, and H9 were detected in <8% of IAV-positive sera.
(SNIP)
DISCUSSION
Pandemic influenza A viruses have historically caused significant mortality and morbidity in humans. These viruses typically originate from animal reservoirs and acquire the capacity for sustained human-to-human transmission through sequential adaptation events 47.
(Continue . . . )
Although this multiplex immunoassay isn't a substitute for PCR testing, it could provide valuable information on the types of viruses circulating in the wild, and levels of herd immunity.
While the last (2009) swine-origin pandemic virus was relatively mild, there are no guarantees that the next one will have similar virulence. Recent studies suggest that community immunity levels to many circulating swine variant viruses are quite limited.
Some recent blogs on swine-origin viruses include:
A reminder that Nature has more than one way at its disposal to spark the next pandemic.
https://afludiary.blogspot.com/2026/04/iscience-multiplex-serological.html
#19,126
While the spillover of HPAI H5N1 into dairy cattle has garnered considerable attention over the past two years, historically, the susceptibility of swine to diverse influenza A viruses has generated the most pandemic concern.
In May of 2023, in Netherlands: Zoonoses Experts Council (DB-Z) Risk Assessment & Warning of Swine As `Mixing Vessels' For Avian Flu, we looked at growing concerns in Europe that avian H5N1 could increase its pandemic threat by spreading (and evolving) in farmed swine.
Only days later, a report out of Italy confirmed an H5N1 spillover event at a `mixed species' farm (poultry & swine), and the subsequent seroconversion of the majority of the pigs tested on that farm (see Study: Seroconversion of a Swine Herd in a Free-Range Rural Multi-Species Farm against HPAI H5N1 2.3.4.4b Clade Virus).
While HPAI detections in swine have been limited, we've seen scattered evidence that H5N1 can infect pigs, albeit often asymptomatically. A few past reports include:
A novel H1N1 virus, circulating in pigs, sparked a pandemic in 2009, and a number of H1, H2, and H3 viruses with zoonotic potential have been identified in swine over the years (see J. Gen. Virology: Evaluation of Pandemic Potential of the Genotype 4 (G4) Swine Influenza Virus).
But testing is extremely limited; PCR testing can only pick up current infection (which may only last days or weeks), while antibody testing (HI/MN/NP-ELISA) can often infer past infections, but are labor intensive and can generally only screen for one subtype at a time.
While RT-PCR can only tell you `what is', and serology only `what was', most countries aren't aggressively testing with either method.
All of which brings us to a new report, published in iScience, which provides a proof-of-concept that a multiplex immunoassay panel can detect a wide range of influenza A subtype antibodies in one pass.
While much of this report will be of interest primarily to lab geeks, the testing took place in Cambodia between 2020 and 2022, which has been the site of a renewed outbreak of HPAI H5N1 since 2023 (see Cambodian MOH Announces 4th Human H5N1 Case of 2026).
While this testing pre-dates this recent resurgence, it revealed a complex panoply of influenza A viruses have circulated in Cambodia pigs, including a faint H5 signal (see graphical abstract below).
The Abstract, and a brief excerpt, from the study follow. I'll have a bit more after the break.
Foong Ying Wong 1 12, Peter Cronin 1 12, Rong Zhang 1 12, Arata Hidano 2 8, Jurre Y. Siegers 3, Dolyce HW Low 1, Hannah Holt 2, William Leung 2, Dina Koeut 4, Bunnary Seng 4, Sovanncheypo Chao 4, Ty Chhay 5, Sothrya Tum 4, San Sorn 4, Monidarin Chou 6, Kimrong Bun 3, Phalla Y 3, Leangyi Heng 3, Marcus G. Mah 1, Giselle GK Ng 1…Yvonne CF Su 1 13 ∗∗Show more
https://doi.org/10.1016/j.isci.2026.115743
Under a Creative Commons license
https://doi.org/10.1016/j.isci.2026.115743
Under a Creative Commons license
Highlights
- Multiplex microsphere immunoassay reveals antibodies to diverse influenza lineages
- H1N1/pdm09 dominates seroprevalence among pigs in Cambodia
- European avian-like swine antibodies indicate ongoing zoonotic risk
- Limited AIV exposure; one pig serum neutralized HPAI H5N1 clade 2.3.4.4b virus
SUMMARY
Animal origin influenza viruses pose significant pandemic threats, with swine serving as key hosts. Serological surveillance in pigs remains limited in regions with intense human–animal–avian contact. Between March 2020 and July 2022, we collected 4,089 pig serum samples in Cambodia, of which 1,321 (32.5%) were influenza A virus (IAV) seropositive by ELISA.
We developed a multiplex microsphere immunoassay comprising a broad panel of hemagglutinin (HA) and neuraminidase (NA) antigens.
Seroprevalence was highest for pandemic H1N1/pdm09 (35.3–45.7%) and lower for classical swine H1 (16.4–17.7%) and European avian-like swine H1 (∼15%) lineages.
Lineage-specific or shared exposures to multiple lineages were observed, indicating complex infection histories. H3 responses varied by clade, while antibodies to avian H5, H7, and H9 were detected in <8% of IAV-positive sera.
These findings underscore ongoing zoonotic risk posed by diverse IAVs circulating in pigs and highlights the need for integrated serological and genomic surveillance.
(SNIP)
DISCUSSION
Pandemic influenza A viruses have historically caused significant mortality and morbidity in humans. These viruses typically originate from animal reservoirs and acquire the capacity for sustained human-to-human transmission through sequential adaptation events 47.
The pandemic H1N1/pdm09 influenza virus exemplifies this process, highlighting the critical role of swine as an intermediate host in the emergence of reassortant strains that had circulated undetected in pigs for nearly a decade before its detection in humans 47,48, largely due to insufficient surveillance.
While genomic surveillance is indispensable for tracing viral origin and identifying emerging variants, multiplex serological assays provide complementary insights by revealing prior exposure histories and population-level immunity in both animal and human hosts.
While genomic surveillance is indispensable for tracing viral origin and identifying emerging variants, multiplex serological assays provide complementary insights by revealing prior exposure histories and population-level immunity in both animal and human hosts.
In this study, we developed a 23-plex microsphere immunoassay targeting a broad diversity of HA and NA antigens representing multiple influenza lineages.
Analysis of NP-positive pig sera collected in Cambodia between 2020 and 2020 demonstrated seroreactivity to a diverse range of H1 lineages, including pandemic H1N1/2009 (pdm09), classical swine (CS), European avian-like (EA) swine and pre-2009 human seasonal lineages. These findings are consistent with our previous genomic surveillance in Cambodia 16, and illustrates the complex multi-lineage influenza ecology within swine populations.
Analysis of NP-positive pig sera collected in Cambodia between 2020 and 2020 demonstrated seroreactivity to a diverse range of H1 lineages, including pandemic H1N1/2009 (pdm09), classical swine (CS), European avian-like (EA) swine and pre-2009 human seasonal lineages. These findings are consistent with our previous genomic surveillance in Cambodia 16, and illustrates the complex multi-lineage influenza ecology within swine populations.
(Continue . . . )
Although this multiplex immunoassay isn't a substitute for PCR testing, it could provide valuable information on the types of viruses circulating in the wild, and levels of herd immunity.
While the last (2009) swine-origin pandemic virus was relatively mild, there are no guarantees that the next one will have similar virulence. Recent studies suggest that community immunity levels to many circulating swine variant viruses are quite limited.
Some recent blogs on swine-origin viruses include:
A reminder that Nature has more than one way at its disposal to spark the next pandemic.
https://afludiary.blogspot.com/2026/04/iscience-multiplex-serological.html