Tembusu virus (TMUV) is a single-stranded, positive-sense RNA virus that belongs to the Flaviviridae
family, Flavivirus genus and Ntaya virus group. The TMUV prototype strain MM1775 (TMUV-MM1775) was first isolated from Culex tritaeniorhynchus
mosquitoes in Kuala Lumpur, Malaysia in 1955. Since 2000, many TMUV strains (TMUVs) have been isolated from birds and mosquitoes, including chicks, ducks, geese, pigeons, sparrows and Culex
mosquitoes. TMUV consists of several genetically closely related virus strains with noticeable pathogenicity for poultry, such as Sitiawan virus (STWV), duck TMUV (DTMUV), Perak virus and Baiyangdian virus [1
]. STWV was isolated from sick broiler chicks in Sitiawan District of Perak State, Malaysia in 2000 [2
]. STWV-infected chicks showed encephalitis, growth retardation and increased blood sugar levels. In 2010, DTMUV, also called duck egg-drop syndrome virus, was found to extensively infect ducks with high morbidity (up to 90%) and mortality (5% to 30%) rates in southeast China [3
]. DTMUV outbreaks also occurred in layer and broiler duck farms in Pekin ducks in Malaysia in 2012 and in Thailand in 2013 [5
The TMUV genome is approximately 11,000 nucleotides and contains 5′ and 3′ untranslated regions (UTRs), and a long open reading frame (ORF) that encodes three structural proteins (capsid (C), pre-membrane protein (prM) and envelope (E)) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Phylogenetic analysis of the nucleotide sequences of the ORFs of several TMUVs showed that TMUV-MM1775 and STWV clustered together away from the genomes of other DTMUVs [7
TMUVs have been isolated from several mosquito species in Asia, mainly Cx. tritaeniorhynchus
] and also Culex
species, including Cx. vishnui
, Cx. gelidus
and Cx. pipiens
]. A mosquito vector competence study by O’Guinn et al. demonstrated that Cx. vishnui
developed high viral titers after feeding on TMUV-infected chicks and could readily transmit TMUV to naive chickens [11
]. Although TMUV are considered mosquito-borne viruses [4
], in vivo studies suggested that TMUVs could be transmitted among ducks by direct contact and aerosol transmission [12
]. The role of mosquitoes as vectors in the transmission of TMUV to avian hosts still needs further study.
Taiwan is an island off the southeastern coast of mainland China that is near the epicenter of DTMUV outbreaks. Taiwan straddles the Tropic of Cancer, having a warm tropical-subtropical climate and large variety of mosquito species and is also an important rest point for migrating birds [14
]. To monitor arboviruses in Taiwan, we conducted a survey on mosquitoes collected from wetlands, pig farms and parks in Taiwan between April and September 2019. In this study, we identified novel TMUV strains in Cx. annulus
and Cx. tritaeniorhynchus
mosquitoes collected from northern and central parts of Taiwan, respectively. We isolated and characterized the genetic sequence of this novel virus.
In this study, we identified two novel TMUV strains, TMUV-TP1906 and TMUV-TC1906, from Cx. annulus
and Cx. tritaeniorhynchus
mosquitoes collected from a wetland in northern Taiwan and a pig farm in central Taiwan, respectively. TMUV-TP1906 was isolated from the Cx. annulus
mosquito pool and grew well in Vero and C6/36 cells without significant cytopathic effects. Through virus isolation and comprehensive genomic and protein sequence analysis, we found that this novel flavivirus is a TMUV-related virus and is most closely related to STWV. STWV, DTMUV, and Bagaza virus, which belong to Ntaya serocomplex virus; and JEV and West Nile virus, which belong to JEV serocomplex virus, have been shown to be primarily Culex
mosquito-associated viruses that cause severe diseases in avian species [7
]. Whether TMUV-TP1906 is a pathogen in birds or other animals needs further study.
Phylogenetic analysis based on the complete ORF of TMUVs showed that TMUVs can be divided into two lineages, TMUV and DTMUV. Analysis of amino acid sequences showed that there are 22 amino acid substitutions unique to the TMUV lineage and 29 amino acid substitutions unique to cluster 1 and cluster 2 of the DTMUV lineage (Figure 4
). The DTMUV-SD14 strain which belongs to cluster 3 of the DTMUV lineage, could be considered as a transitional group between the TMUV lineage and clusters 1 and 2 of the DTMUV lineage. The DTMUV-SD14 strain contains 48 amino acid mutations, which are different from other TMUVs. In addition, there are five informative amino acid substitutions (NS1-105, NS1-205, 2K-13, NS4B-30 and NS5-562) for the differentiation of the TMUV lineage, DTMUV-SD14 (cluster 3 of the DTMUV lineage) and clusters 1 and 2 of the DTMUV lineage. The TMUV lineage contains three virus strains, the TMUV-MM1775 prototype strain, STWV, and TMUV-TP1906. TMUV-MM1775 has not been documented as a pathogen in birds or other animals; however, STWV, which is most closely related to TMUV-TP1906, causes encephalitis and retards the growth of chicks. Analysis of amino acid sequences showed that TMUV-TP1906 contains 12 unique amino acid substitutions compared with other TMUVs. In addition, comparison of protein sequences of TMUV-TP1906 and STWV with other TMUVs showed 10 amino acid substitutions. These informative amino acid positions may be useful for the classification of TMUVs and may play crucial roles in the evolution and protein function of TMUVs.
The Asn glycosylation site at residue 154 of the E protein (154-Asn) of TMUVs is critical for virus tissue tropism and transmissibility in poultry [24
]. TMUV-MM1775 contains 156-Pro, which can disrupt the N-linked glycosylation of 154-Asn of the E protein, and thus may affect viral virulence and transmissibility in avian species [24
]. Except for TMUV-MM1775, all the other TMUVs, including TMUV-TP1906, contain 156-Ser which would not disrupt 154-Asn glycosylation and thus may not affect the glycosylation-associated infectivity of TMUVs in avian species. A recent study showed that the Thr to Lys mutation at residue 367 of the E protein plays a predominant role in viral cell-adaptation and virulence attenuation in ducks [25
]. TMUV-TP1906 contains 367-Thr of the E protein and thus may retain the virulence in the host. In addition to E protein, NS1 protein contains three N-linked glycosylation sites at residues 130, 175, and 207. A recent study showed that some DTMUV strains had a glycosylation motif mutation at residues 175–178 from NTTD to NITD but the glycosylation site at 130 was conserved [19
]. Interestingly, we found NS1 glycosylation motif at residues 175 and 207 were conserved in TMUV-TP1906 and TMUV-TC1906, however, TMUV-TC1906 had a glycosylation motif mutation at residues 130–133 from NNTF to NSTF. Whether these mutations affect protein functions or virulence needs further study.
The 5′ and 3′ UTRs of flaviviruses are important for virus replication and transmission [26
]. The 5′ end of the 3′UTR of flavivirus is known to have a VR with different nucleotide lengths [26
], and this VR sequence may be associated with the production of subgenomic flavivirus RNA and immune modulation, hence contributing to virus adaptation in vector and non-vector hosts [27
]. The 3′UTR VRs of TMUVs consist of 84 to 94 nucleotides immediately downstream of the ORF. Phylogenetic analysis of 3′UTR VR and ORF sequences showed similar topological features, indicating that 3′UTR VRs may serve as a potential marker for TMUV evolution. Sequence analysis also showed that the 3′UTR VR of TMUV-TP1906 is very unique among TMUVs (Figure 5
). More research is needed to understand the genetic diversity and function of the 3′UTR VR of TMUVs.
Prominent CPE was observed in BHK-21, C6/36, Vero and DF-1 cell lines after infection with DTMUV [5
]. STWV infection induced CPE in the BK3 cells (chicken bursal lymphoma cell line) but not in CPK (porcine kidney cell line), MARC-145 (monkey kidney cell line) and Vero cells [2
]. In our study, we found that the TMUV-TP1906, which is closely related to STWV, grew in C6/36 and Vero cell without inducing significant CPE, however, the virus caused drastic CPE in DF-1 and BHK-21 cell lines (Figure 2
TMUVs are emerging pathogenic flaviviruses causing severe avian diseases in Malaysia, Thailand, and China. Taiwan is located south of East Asia and is close to the epicenter of DTMUV outbreaks in China. In this study, we first identified a TMUV in Taiwan. Interestingly, TMUV-TP1906 is most closely related to STWV and TMUV-MM1775 found in Malaysia and is different from the DTMUVs found in China (Figure 3
and Figure 4 Figure 3
; Figure 4
). Previous study has divided TMUVs into the Chinese mainland TMUV lineage and Southeast Asian TMUV lineage [8
]. According to the phylogeny, TMUV-MM1775, STWV, TMUV-TP1906, and two TMUV strains identified from Cx. tritaeniorhynchus
in Yunnan Province of China were grouped as the Southeast Asian TMUV lineage. The phylogeographical analysis suggested that the TMUVs might spread from Southeast Asian countries such as Malaysia and Thailand to the Yunnan Province of China (southern China) and further spread to areas in northern China such as Shandong Province. However, the mechanism of long-distance spread of TMUVs is still unknown. Liu et al. suggested that the spread of TMUVs might occur via migratory birds and ornithophilic Culex
spp. mosquitoes, since TMUVs have been identified in wild birds such as pigeons and mallards (Anas platyrhynchos
]. The East Asian–Australasian flyway is one of the world’s great flyways for migratory birds, and the flyway passes through many countries including Malaysia, Thailand, China, and Taiwan; thus, it is possible that the spread of TMUVs might be through this flyway. In addition, it has been suggested that the spread of avian influenza virus and JEV might also occur through this flyway [30
In this study, we reported the first isolation of a novel TMUV strain from Culex mosquitoes in Taiwan. In addition, it is the first time that the TMUV strain was isolated from Cx. annulus mosquitoes. We performed comprehensive genomic and amino acid analyses between TMUV-TP1906 and other TMUVs, and our results may help to increase understanding of the diversity and evolution of TMUVs. Further study is warranted to investigate the virulence and epidemiology of TMUV-TP1906 in Taiwan.