Duck farming is a long-standing agricultural venture in Asia. However, this sector faces challenges from various infectious agents, including duck Circovirus, Sitiawan virus, and duck Tembusu virus (TMUV) (1–3). TMUV, classified within the genus Flavivirus of the Flaviviridae family, is an arthropod-borne virus characterized by a single-stranded, positive-sense RNA genome. It was originally isolated in 1955 from Culex tritaeniorhynchus mosquitoes in Kuala Lumpur, Malaysia (3). Nevertheless, its potential implications for human and animal health remain incompletely understood. Within the Flavivirus genus, several members, such as West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and Zika virus, exhibit zoonotic properties and serve as major vector-borne pathogens responsible for millions of annual infections. These infections can manifest with a spectrum of clinical presentations, ranging from mild febrile symptoms to severe and potentially fatal hemorrhagic or neurologic diseases. This viral genus is characterized by its 30–60 nm icosahedral envelope capsid containing a single-stranded positive-sense RNA genome approximately 11 Kb in length. The genomic organization encoding the singular polyprotein is three structural proteins of capsid (C)-premembrane (prM)-envelope (E) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) that are produced through the actions of viral and cellular proteases. Surrounding the coding region are untranslated regions at both the 5′ and 3′ ends, which form conserved stem-loop structures (3, 4). Primarily, the composition of the viral particle is shaped by the structural proteins, which play a pivotal role in mediating viral fusion with host cells. These proteins are essential in various processes, including binding to virus receptors, facilitating entry, and associating fusion events. In contrast, the non-structural (NS) proteins are primarily engaged in activities, such as viral RNA replication, the assembly of virions, and the evasion of innate immune responses (5).

Within natural ecosystems, avian species are reservoir hosts for a multitude of flaviviruses, such as West Nile virus (WNV) (6), Sitiawan virus (3), Usutu virus (7, 8), and Bagaza virus (9). In April 2010, the causative agent of duck egg-drop disease in China was first recognized as duck TMUV. This ailment is distinguished by a decrease in egg yield, an abrupt reduction in feed intake, and the emergence of neurological manifestations among afflicted egg-laying and breeder ducks (4). Egg-drop disease impacts a diverse array of duck breeds, encompassing both meat-producing and egg-laying categories. This spectrum includes Pekin ducks, Cherry Valley ducks, Shaoxing ducks, Jinyun ducks, Longyan ducks, Jinding ducks, Khaki-Campbell ducks, Muscovy ducks, and domesticated mallards (10). The virus is still circulating and affecting duck production and many countries. It would be further studied on epidemiological and genetic characterization of the viral strains, which gives important information for developing vaccine and preventive strategies.

In Vietnam, the first study on TMUV infection was reported by Dang et al. Those authors used a PCR method to detect the TMUV genome in clinically suspected ducks in Hanoi city. Phylogenetic analysis of the partial NS5B gene sequences suggested that the three Vietnamese TMUV strains were genetically related to the DK/TH/CU-1 strain (KR061333) detected in Thailand (11). The aim of the current study was to carry out further epidemiological and genetic characterization of the TMUV strains circulating in several cities and provinces in north Vietnam based on their partial E and prM gene sequences.

It has been reported that TMUV was successfully isolated from mosquitoes in Malaysia in 1950s (17). Subsequently, the virus spread and has been reported in many countries globally (1, 2, 4, 7, 11, 18). Understanding infection is important in creating strategies to control the disease. In Vietnam, the first report of TMUV infection was conducted in 2019 (11). The current study has continued to describe viral infection among ducks raised in some provinces in north Vietnam. Epidemiological analysis was first conducted in this study, with the five novel Vietnamese TMUV strains obtained in this study genetically forming a single cluster (2b), separated from the vaccine strain based on the partial E gene sequences (891 bp). This finding suggested that these strains were field strains.

Another study noted that TMUV infection could reach 90% among ducks within a farm, while the mortality rate varied from 5 to 30% (19). In the current study, The rates for TMUV-positive samples and farms were 16.15 and 23.68%, respectively, based on PCR. These rates were lower than the rate of 46.59% (41/88) reported in China during 2010–2016 (20) due to the fact that the sampling number, location, and time parameters were different, resulting in differences in the infection rates. Li et al. conducted a study to access pathogenicity of TMUV strains isolated in China (21). One-, 3-, and 7-weeks-old ducks were inoculated with TMUV strains. The study pointed out that ducks at one-week-old indicated severe symptoms, while other groups showed milder pathological lesions (21). This finding agreed with a previous study revealed that young ducks were more sensitive to TMUV strains than older ducks, suggesting a relationship between viral susceptibility and age (21, 22). Later researchers found that ducks aged 18–21 weeks and 55 weeks were susceptible to the disease caused by TMUV (23, 24). Changes in age-related susceptibility occurred among ducks infected with TMUV strains. It is due to the fact that host immune responses, different viral strains, or evolution among viral strains may contribute to age-related susceptibility. Further studies should be conducted to elucidate these points. The current study indicated that the highest rate of viral infection was in young ducks aged 2–4 weeks (59.26%), compared to older ducks aged >4 weeks (5.43%), with no positive samples being detected in ducks aged <2 weeks. Older ducks may have a stronger immune response, leading to lower positive rates (21). Further studies should be conducted to explain the relationship between age and infection rates in north Vietnam.

Genetic and phylogenetic analyses based on the partial E and prM gene sequences revealed that the current five Vietnamese TMUV strains formed a novel subcluster, which was closely related to Chinese strains. The results suggested that the TMUV strains obtained in this study might have a similar origin to Chinese strains. N-linked glycosylation, which contributes to the entry of the virus into host cells, was predicted in the E protein of TMUV at the 103, 154, and 314 residues (25, 26). However, in the current study, substitutions were not found at these three residues, suggesting the conservation of the glycosylation sites of E protein among Vietnamese TMUV strains, which was similar to the findings of Huang et al. Fritz et al. suggested that protonation of histidine’s plays an important role in the membrane fusion of flaviviruses (27). No substitutions of histidine was observed at the 144, 153, 163, 219, 246, 263, 285, 320, and 398 residues of the E protein of the five Vietnamese TMUVs in the current study, nor were substitutions found in the epitopes 220–226 and the 374–380 residues, located in domains DII and DIII in the E protein of the Vietnamese TMUV strains. In the current study, three substitutions were found among Vietnamese TMUV strains: residues 110 (K → E), 157 (A → V), and 345 (D → N). To our knowledge, no evidence on the contribution of these substitutions has been reported; other studies will be conducted to evaluate this point.

One of the evolutionary processes is recombination, was first found among TMUV strains in China (28). The current study did not find any recombination events among the Vietnamese TMUV strains based on analysis of the partial S gene. Expanding analysis of the complete genome and increasing the number of sequences are necessary to examine possible recombination events among Vietnamese TMUV strains. Dai et al. (16) reported that negative selection was strong among Chinese TMUV strains. A few positive selection sites were found among the TMUV strains as a result of genetic drifts, similar to other flaviviruses (29, 30). In the current study, 169 residues of Vietnamese TMUV E protein had negative selection.

In this study, TMUV infection in samples and on duck farms were 16.15 and 23.68%, respectively, in six provinces/city in north Vietnam in 2021. The infection was most commonly detected in young ducks aged 2–4 weeks (59.26%), at a significantly higher level than in ducks aged <2 weeks and > 4 weeks. Genetic and phylogenetic analyses of the five Vietnamese TMUV strains based on the partial E and prM gene sequences supported that the current Vietnamese TMUV strains belonged to a novel subcluster, which was closely related to the Chinese strains and differed from the vaccine strain. No putative recombination event was detected among the Vietnamese TMUV strains. Strong negative selection was found among the Vietnamese TMUV strains, based on the analysis of the partial E protein. Further studies should be conducted to better understand the evaluation of TMUV strains across the country.

The original contributions presented in the study are publicly available. This data can be found here: https://www.ncbi.nlm.nih.gov/nuccore/; OR727885-OR727894.

The animal studies were approved by the Committee on Animal Research and Ethics of the University (CARE-2021/04). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

HD: Conceptualization, Methodology, Software, Writing – original draft, Data curation, Formal analysis, Investigation, Visualization. GT: Conceptualization, Writing – original draft, Data curation, Formal analysis, Investigation, Visualization. TV: Writing – original draft. NL: Writing – original draft. YN: Writing – original draft. WR: Writing – original draft. AR: Writing – original draft. CB: Writing – original draft. DB: Writing – original draft, Conceptualization, Software, Supervision, Writing – review & editing. JR: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Software, Supervision, Validation, Writing – original draft, Writing – review & editing.