Chinese researchers have isolated and characterized a new Tembusu virus strain from a laying hen farm in Hubei Province, China, where an outbreak in 2025 caused depression, paralysis, and a marked decline in egg production among affected birds. The isolate, designated CHN-WH, belongs to genetic cluster 3.2 of this emerging avian flavivirus and carries a unique amino acid substitution in its envelope protein that distinguishes it from all other reference strains analyzed. In experimental infections, the virus caused systemic disease with neurological lesions in young chicks and ovarian damage with sharply reduced egg output in laying hens, according to a study published in the journal Animal Diseases.
Tembusu virus is an arbovirus of the genus Orthoflavivirus within the family Flaviviridae, first isolated in 1955 from Culex tritaeniorhynchus mosquitoes. The first recognized TMUV-associated disease in poultry was reported in 2000 in chicken flocks in Malaysia, characterized by encephalitis, growth retardation, and hyperglycemia. In April 2010, a severe outbreak struck major waterfowl-producing regions of China, causing marked reductions in egg production and pronounced neurological disorders in ducks and substantial economic losses to the duck industry. Since then, the virus has become widely distributed and has caused recurrent epidemics in duck flocks in China and several Southeast Asian countries. Subsequent studies have shown that TMUV can infect a broad range of avian species, including quails, geese, pigeons, and sparrows, and fatal natural infection has more recently been documented in Indo-Pacific bottlenose dolphins in Thailand, providing the first evidence of natural mammalian disease.
The investigation began when ovarian tissue samples were collected from laying hens with suspected TMUV infection on the affected farm. Reverse transcription PCR testing revealed TMUV positivity, whereas screening for other common avian viruses, including infectious bronchitis virus, avian influenza virus, Newcastle disease virus, egg drop syndrome virus, and Marek’s disease virus, yielded negative results. When TMUV-positive samples were inoculated into 10-day-old specific-pathogen-free duck embryos, typical embryo death accompanied by hemorrhages was observed. BHK-21 cells subsequently inoculated with TMUV-positive allantoic fluid developed pronounced cytopathic effects characterized by cell shrinkage and detachment. The isolate was confirmed by an immunofluorescence assay using a monoclonal antibody specific for the TMUV envelope protein, and similar fluorescence patterns were observed in infected chicken embryo fibroblasts and duck embryo fibroblasts. Transmission electron microscopy of purified virions revealed spherical particles 40 to 60 nanometers in diameter, consistent with the morphology of flaviviruses.
Growth curve experiments demonstrated that CHN-WH replicated efficiently in all three cell types tested. The virus reached peak titers of 10^7.46 TCID50 per milliliter in BHK-21 cells at 36 hours post infection, 10^7.49 TCID50 per milliliter in chicken embryo fibroblasts at 48 hours post infection, and 10^7.29 TCID50 per milliliter in duck embryo fibroblasts at 48 hours post infection. This broad replication capacity in cells derived from both chicken and duck embryos aligns with the view that cluster 3.2 strains possess broader biological adaptability than some other TMUV lineages.
Whole-genome sequencing showed that CHN-WH possesses a genome of 10,990 bases containing a single open reading frame between nucleotides 96 and 10,372, encoding a polyprotein of 3,425 amino acids. The sequence has been deposited in GenBank under accession number PX630769.1. Sequence alignment revealed that CHN-WH shared between 87.69 and 99.34 percent nucleotide identity with the other 60 TMUV strains included in the analysis. A maximum-likelihood phylogenetic tree constructed from complete open reading frame sequences divided the strains into five distinct clusters, designated clusters 1, 2.1, 2.2, 3.1, and 3.2. CHN-WH was assigned to cluster 3.2 and showed the closest genetic relationship to the mosquito-derived strain TMUV-YN2020, sharing 99.34 percent nucleotide identity. Clusters 2.1 and 2.2 contain mainly classical waterfowl-origin strains associated with large-scale outbreaks of egg-drop syndrome and neurological disease in ducks, whereas cluster 3.2 includes genetically diverse isolates from mosquitoes, geese, chickens, ducks, sparrows, pigeons, quails, and dolphins, with an increasing number of isolates reported in recent years.
Comparative analysis of the envelope protein, the major surface glycoprotein that mediates viral attachment, membrane fusion, tissue tropism, and antigenicity, revealed several distinctive molecular features. CHN-WH carried E52 and P83 rather than the D52 and S83 residues characteristic of cluster 1 strains, and carried D89, L180, A312, and S332 instead of the E89, M180, V312, and T332 residues characteristic of clusters 2.1 and 2.2. In addition, the virus carried eight amino acid signature residues, namely S69, V91, I135, N150, I358, P365, V371, and L493, that are characteristic of cluster 3.2 strains and absent from representative strains of the other clusters. Structural modeling indicated that the envelope protein retained the typical flavivirus architecture of three ectodomains and a C-terminal transmembrane region, with the signature residues distributed across these domains. Notably, CHN-WH was the only analyzed strain carrying a leucine at residue 198, whereas all other reference strains retained a conserved methionine at this position. This M198L substitution was predicted to be surface exposed and located near the domain II fusion loop, although the researchers emphasized that its functional relevance remains unknown and requires experimental validation.
To assess pathogenicity in young birds, 5-day-old specific-pathogen-free chicks were inoculated intramuscularly with 10^6 TCID50 of the virus, while control chicks received an equal volume of culture medium. Infected chicks developed depression, reduced food intake, and vent pecking, accompanied by significantly reduced body weight gain. The maximum mean daily gain was 28.0 grams in infected chicks compared with 47.8 grams in control chicks. Mortality began at 9 days post infection, and the survival rate of the infected group was 87.5 percent. Gross examination at 3 days post infection revealed cerebral hemorrhage, splenomegaly, and thymic hemorrhage with enlargement. Histopathological analysis showed lesions in the brain, spleen, and thymus, including perivascular cuffing, satellitosis, and neuronophagia in the brain, lymphocytic necrosis and apoptosis in the spleen, and cortical atrophy with lymphocyte apoptosis in the thymus.
Virological measurements confirmed systemic dissemination in the infected chicks. Viremia peaked at 3 days post infection with a maximum viral RNA load of 6.4 log10 copies per milliliter in blood and became undetectable by 14 days post infection. The virus replicated efficiently in multiple organs, including the heart, liver, spleen, lung, kidney, brain, thymus, bursa of Fabricius, pancreas, duodenum, and yolk sac, with peak tissue viral RNA loads ranging from 4.95 to 6.95 log10 copies per gram. No viral RNA was detected in any blood or tissue samples from the mock-infected group throughout the experiment. In laying hens, 30-week-old birds inoculated subcutaneously with 10^6 TCID50 developed splenomegaly and ovarian follicular hemorrhage. Egg production gradually declined after infection, from 80 percent before infection to 33 percent at 7 days post infection, a drop of 47 percentage points. Histopathological examination of the ovary revealed follicular deformation, granulosa cell vacuolization, and follicular atresia, while the spleen showed lymphocytic nuclear pyknosis consistent with necrosis or apoptosis. Viral RNA was detected in both the spleen and ovary, with mean loads of 5.55 and 5.31 log10 copies per gram, respectively, indicating active viral replication in these tissues.
The findings have implications for disease control because currently available TMUV vaccines are derived primarily from cluster 2 strains, and no vaccines specifically targeting cluster 3.2 viruses are available. A licensed live-attenuated cluster 2 vaccine induced high antibody levels in chickens and prevented viral dissemination following challenge with the chicken-origin cluster 3.2 strain SD2021, but other studies have identified substantial antigenic differences between cluster 3 viruses and representative cluster 2.2 strains, suggesting that protection conferred by cluster 2-based vaccines against contemporary cluster 3.2 viruses may be partially or strain dependent. The authors also noted important limitations: a parallel mock-infected control group was not included in the egg-production experiment, so the observed decline should be interpreted relative to the preinfection baseline, and the relatively small sample size used for necropsy and viral load analyses warrants caution. Direct-contact transmission among chickens has not been demonstrated for chicken-origin cluster 3.2 strains under tested conditions, and the transmission characteristics of CHN-WH require further experimental evaluation. The researchers suggest that CHN-WH provides a relevant contemporary isolate for future assessments of vaccine matching, cross-neutralization, and protective efficacy against cluster 3.2 strains circulating in chickens.
Subject of Research: Genomic and pathogenic characterization of a chicken-origin cluster 3.2 Tembusu virus strain isolated from a laying hen farm in China
Article Title: Genomic and pathogenic characterization of a chicken-origin cluster 3.2 Tembusu virus isolated in China
Article References: Chen, D., Hu, S., Zhang, Y., Jin, H., Chen, S., Zhou, H., Yi, C., Han, Y., Zhou, P., & Luo, R. (2026). Genomic and pathogenic characterization of a chicken-origin cluster 3.2 Tembusu virus isolated in China. Animal Diseases, 6(1), Article 50. https://doi.org/10.1186/s44149-026-00264-4
Image Credits: AI Generated
DOI: 10.1186/s44149-026-00264-4
Keywords: Tembusu virus, flavivirus, cluster 3.2, poultry, laying hens, egg production, encephalitis, viral pathogenicity, E protein, phylogenetics, China, vaccine development
News Source: Kristina Jarvis. (October 29, 2026). New Chicken-Origin Tembusu Virus Strain Shows Distinct Genes and Pathogenicity in China. Scienmag.
