Objective:

To investigate mosquito-associated flavivirus diversity in Vietnam, determine whether Tembusu virus (TMUV)–related RNA could be detected in field-caught mosquitoes, and place any recovered sequence in regional phylogenetic context.

Approach:

• Mosquito Collection: Mosquitoes were collected at 12 sites representing urban, rural, and forested habitats across northern, southern, Central Coast, and Central Highlands regions of Vietnam during 2022–2023.
• Study Sample: Of 18,091 mosquitoes collected, 10,658 female Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Culex tritaeniorhynchus mosquitoes were included and grouped into 586 pools.
• Molecular Screening: Pools were screened for flaviviruses and alphaviruses using broad-range RT-PCR. Flavivirus-reactive amplicons underwent Sanger sequencing.
• Genomic and Phylogenetic Analysis: One pool with a TMUV-like signal underwent viral metagenomic sequencing. Recovered genomic regions were compared with publicly available TMUV sequences from mosquito and avian hosts in Asia.

Key Findings:

• Nine pools produced bands of the expected size on pan-flavivirus RT-PCR, but Sanger sequencing did not definitively identify viral sequences from these pools.
• One pool containing 25 Cx tritaeniorhynchus mosquitoes collected at a rural site in southern Vietnam yielded a TMUV draft genome after deeper sequencing.
• The recovered sequence comprised 6,764 bases, representing 61.5% of the complete genome.
• The Vietnamese mosquito-derived sequence clustered within a distinct monophyletic clade containing TMUV strains from China, Thailand, Taiwan, and Vietnam.
• This regional clade predominantly included poultry-associated sequences, together with 2 mosquito-derived sequences and 1 sequence associated with a bottlenose dolphin.
• The Vietnamese sequence had 98.44% nucleotide similarity to 2 Chinese sequences derived from mosquito and chicken hosts and formed a monophyletic group with a goose-associated sequence from China.
• No alphavirus-positive bands or additional arbovirus sequences relevant to the study objective were identified.

Interpretation:

The recovered viral RNA provides the first mosquito-based molecular evidence of a TMUV-related virus in Vietnam and places it within a broader regional clade containing mosquito- and poultry-associated strains. However, RNA detection does not confirm an infectious virus or demonstrate that Cx tritaeniorhynchus can transmit TMUV. The available phylogenetic data also cannot determine the direction or timing of regional spread.

Limitations:

• Detection of viral RNA in field-caught mosquitoes did not establish the presence of infectious virus or mosquito vector competence.
• Most pan-flavivirus RT-PCR–reactive bands could not be confirmed by Sanger sequencing and may have reflected low RNA concentrations, degradation, or nonspecific amplification.
• The recovered genome was incomplete and covered less than 70% of the full genome, limiting detailed genomic comparisons and assessment of mutations.
• Mosquito species and geographic regions were unevenly represented.
• Sampling was not designed to estimate TMUV prevalence, geographic distribution, seasonality, or vector competence.
• The survey may not have captured spatial, seasonal, annual, or habitat-specific variation in mosquito populations and viral circulation.

Conclusion:

A TMUV-related draft genome was recovered from a pool of Cx tritaeniorhynchus mosquitoes collected in rural southern Vietnam, providing the country’s first mosquito-based molecular evidence of the virus. Longitudinal mosquito surveillance combined with sampling of ducks, wild birds, and poultry farms, TMUV-specific assays, virus isolation, and complete-genome sequencing is needed to clarify its epidemiology, genetic diversity, and transmission ecology.