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ABSTRACT: Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. Duck Tembusu virus genome encodes one polyprotein that undergoes cleavage to produce 10 proteins. Among these, NS4B, the largest transmembrane protein, plays a crucial role in the viral life cycle. In this study, we investigated the localization of NS4B and found that it is located in the endoplasmic reticulum, where it co-localizes with DTMUV dsRNA. Subsequently, we confirmed 5 different transmembrane domains of NS4B and discovered that only its transmembrane domain 3 (TMD3) can traverse ER membrane. Then mutations were introduced in the conserved amino acids of NS4B TMD3 of DTMUV replicon and infectious clone. The results showed that V111G, V117G, and I118G mutations enhanced viral RNA replication, while Q104A, T106A, A113L, M116A, H120A, Y121A, and A122G mutations reduced viral replication. Recombinant viruses with these mutations were rescued and studied in BHK21 cells. The findings demonstrated that A113L and H120A mutations led to higher viral titers than the wild-type strain, while Q104A, T106A, V111G, V117G, and Y121A mutations attenuated viral proliferation. Additionally, H120A, M116A, and A122G mutations enhanced viral proliferation. Furthermore, Q104A, T106A, V111G, M116A, V117G, Y121A, and A122G mutants showed reduced viral virulence to 10-d duck embryos. Animal experiments further indicated that all mutation viruses resulted in lower genome copy numbers in the spleen compared to the WT group 5 days postinfection. Our data provide insights into the topological model of DTMUV NS4B, highlighting the essential role of NS4B TMD3 in viral replication and proliferation.

Since its outbreak in 2010, Tembusu virus (TMUV) has spread widely throughout China and Southeast Asia, causing significant economic losses to the poultry industry. In 2018, an attenuated vaccine called FX2010-180P (180P) was licensed for use in China. The 180P vaccine has demonstrated its immunogenicity and safety in mice and ducks. The potential use of 180P as a backbone for flavivirus vaccine development was explored by replacing the pre-membrane (prM) and envelope (E) genes of the 180P vaccine strain with those of Japanese encephalitis virus (JEV). Two chimeric viruses, 180P/JEV-prM-E and 180P/JEV-prM-ES156P with an additional E protein S156P mutation were successfully rescued and characterized. Growth kinetics studies showed that the two chimeric viruses replicated to similar titers as the parental 180P virus in cells. Animal studies also revealed that the virulence and neuroinvasiveness of the 180P/JEV-prM-E chimeric virus was decreased in mice inoculated intracerebrally (i.c.) and intranasally (i.n.), respectively, compared to the wild-type JEV strain. However, the chimeric 180P/JEV-prM-E virus was still more virulent than the parent 180P vaccine in mice. Additionally, the introduction of a single ES156P mutation in the chimeric virus 180P/JEV-prM-ES156P further attenuated the virus, which provided complete protection against challenge with a virulent JEV strain in the mouse model. These results indicated that the FX2010-180P could be used as a promising backbone for flavivirus vaccine development. [ABSTRACT FROM AUTHOR]

ABSTRACT Tembusu virus (TMUV), an avian mosquito-borne flavivirus, was first identified from Culex tritaeniorhynchus in 1955. To validate the effects of the 3′-untranslated region (3′UTR) in viral host-specific adaptation, we generated a set of chimeric viruses using CQW1 (duck strain) and MM 1775 (mosquito strain) as backbones with heterogeneous 3′UTRs. Compared with rMM 1775, rMM-CQ3′UTR (recombinant MM 1775 virus carrying the 3′UTR of CQW1) exhibited enhanced proliferation in vitro, with peak titers increasing by 5-fold in duck embryonic fibroblast (DEF) cells or 12-fold in baby hamster kidney (BHK-21) cells; however, the neurovirulence of rMM-CQ3′UTR was attenuated in 14-day-old Kunming mice via intracranial injection, with slower weight loss, lower mortality, and reduced viral loads. In contrast, rCQ-MM3′UTR showed similar growth kinetics in vitro and neurovirulence in mice compared with those of rCQW1. Then, the Stem-loop I (SLI) structure, which showed the highest variation within the 3′UTR between CQW1 and MM 1775, was further chosen for making chimeric viruses. The peak titers of rMM-CQ3′UTRSLI displayed a 15- or 4-fold increase in vitro, and the neurovirulence in mice was attenuated, compared with that of rMM 1775; rCQ-MM3′UTRSLI displayed comparable multiplication ability in vitro but was significantly attenuated in mice, in contrast with rCQW1. In conclusion, we demonstrated that the TMUV SLI structure of the 3′UTR was responsible for viral host-specific adaptation of the mosquito-derived strain in DEF and BHK-21 cells and regulated viral pathogenicity in 14-day-old mice, providing a new understanding of the functions of TMUV 3′UTR in viral host switching and the pathogenicity changes in mice. IMPORTANCE Mosquito-borne flaviviruses (MBFVs) constitute a large number of mosquito-transmitted viruses. The 3′-untranslated region (3′UTR) of MBFV has been suggested to be relevant to viral host-specific adaptation. However, the evolutionary strategies for host-specific fitness among MBFV are different, and the virulence-related structures within the 3′UTR are largely unknown. Here, using Tembusu virus (TMUV), an avian MBFV as models, we observed that the duck-derived SLI of the 3′UTR significantly enhanced the proliferation ability of mosquito-derived TMUV in baby hamster kidney (BHK-21) and duck embryonic fibroblast (DEF) cells, suggesting that the SLI structure was crucial for viral host-specific adaptation of mosquito-derived TMUVs in mammalian and avian cells. In addition, all SLI mutant viruses exhibited reduced viral pathogenicity in mice, indicating that SLI structure was a key factor for the pathogenicity in mice. This study provides a new insight into the functions of the MBFV 3′UTR in viral host switching and pathogenicity changes in mice.

ABSTRACT: Flavivirus RNA cap-methylation plays an important role in viral infection, proliferation, and escape from innate immunity. The methyltransferase (MTase) of the flavivirus NS5 protein catalyzes viral RNA methylation. The E218 amino acid of the NS5 protein MTase domain is one of the active sites of flavivirus methyltransferase. In flaviviruses, the E218A mutation abolished 2’-O methylation activity and significantly reduced N-7 methylation activity. Tembusu virus (TMUV, genus Flavivirus) was a pathogen that caused neurological symptoms in ducklings and decreased egg production in laying ducks. In this study, we focused on a comprehensive understanding of the effects of the E218A mutation on TMUV characteristics and the host immune response. E218A mutation reduced TMUV replication and proliferation, but did not affect viral adsorption and entry. Based on a TMUV replicon system, we found that the E218A mutation impaired viral translation. In addition, E218A mutant virus might be more readily recognized by RIG-I-like receptors to activate the corresponding antiviral immune signaling than WT virus. Together, our data suggest that the E218A mutation of TMUV MTase domain impairs viral replication and translation and may activates RIG-I-like receptor signaling, ultimately leading to a reduction in viral proliferation.

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. Duck Tembusu virus genome encodes one polyprotein that undergoes cleavage to produce 10 proteins. Among these, NS4B, the largest transmembrane protein, plays a crucial role in the viral life cycle. In this study, we investigated the localization of NS4B and found that it is located in the endoplasmic reticulum, where it co-localizes with DTMUV dsRNA. Subsequently, we confirmed 5 different transmembrane domains of NS4B and discovered that only its transmembrane domain 3 (TMD3) can traverse ER membrane. Then mutations were introduced in the conserved amino acids of NS4B TMD3 of DTMUV replicon and infectious clone. The results showed that V111G, V117G, and I118G mutations enhanced viral RNA replication, while Q104A, T106A, A113L, M116A, H120A, Y121A, and A122G mutations reduced viral replication. Recombinant viruses with these mutations were rescued and studied in BHK21 cells. The findings demonstrated that A113L and H120A mutations led to higher viral titers than the wild-type strain, while Q104A, T106A, V111G, V117G, and Y121A mutations attenuated viral proliferation. Additionally, H120A, M116A, and A122G mutations enhanced viral proliferation. Furthermore, Q104A, T106A, V111G, M116A, V117G, Y121A, and A122G mutants showed reduced viral virulence to 10-d duck embryos. Animal experiments further indicated that all mutation viruses resulted in lower genome copy numbers in the spleen compared to the WT group 5 days postinfection. Our data provide insights into the topological model of DTMUV NS4B, highlighting the essential role of NS4B TMD3 in viral replication and proliferation. [ABSTRACT FROM AUTHOR]

The disease caused by the Tembusu virus (TMUV) is characterized by egg-drop syndrome in ducks. In recent years, TMUV has become a concern due to the expansion of host species and increasing number of infection records in Asia. This study aimed to develop a sensitive and rapid reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method to target highly conserved regions using a colori fluorescent indicator (CFI) for TMUV detection. Two primer sets for RT-LAMP, including loop primers, were designed from the conserved region of the NS5 gene to accelerate amplification efficacy. The specificity and sensitivity of the RT-LAMP method were evaluated using the TMUV FX2010-180P vaccine strain and other duck-related viruses. RT-PCR and RT-LAMP concordance was evaluated based on results of 64 clinical samples. The developed RT-LAMP method was specific for TMUV. The sensitivity of the RT-LAMP method was 100 times higher than that of the two-step RT-PCR. The concordance between RT-PCR and RT-LAMP was 92.19%. A reliable, rapid, and simple RT-LAMP method for TMUV detection was successfully developed. This is the first RT-LAMP-based TMUV detection method with the addition of two loop primers for higher sensitivity and rapidity that could provide a potential strategy for screening TMUV infection. [ABSTRACT FROM AUTHOR]

Flavivirus envelope protein (E) plays an important role in cellular infection, especially in virulence and antigenicity. E domain III of Tembusu virus (TMUV) is highly conserved among flaviviruses and contains four loop regions. However, the functions of the loop regions of TMUV E domain III in the viral life cycle have not yet been discovered. In this study, using a reverse genetics system, we performed site-directed mutagenesis on loops I, II, III, and IV of TMUV E domain III. Mutant 6 (S388A.G389A.K390A) showed better proliferation than the wild-type virus, while mutants 1–5 exhibited decreased in vitro infectivity, as determined by immunofluorescence assay (IFA). Based on a TMUV replicon system, the mutations exhibited no apparent effect on TMUV RNA replication. Subcellular fractionation assays and packaging system assays indicated that mutations in loops II–IV (T332A, T332S, S365A.S366A.T367A, and S388A.G389A.K390A, respectively) disrupted virion assembly. Moreover, loops I–IV played an important role in virus binding and entry, while mutant 6 (S388A.G389A.K390A) exhibited robust activity in virus entry. Taken together, our findings indicated the critical role of the loop regions in TMUV E domain III in the virus entry and assembly process.

Flavivirus envelope protein (E) plays an important role in cellular infection, especially in virulence and antigenicity. E domain III of Tembusu virus (TMUV) is highly conserved among flaviviruses and contains four loop regions. However, the functions of the loop regions of TMUV E domain III in the viral life cycle have not yet been discovered. In this study, using a reverse genetics system, we performed site-directed mutagenesis on loops I, II, III, and IV of TMUV E domain III. Mutant 6 (S388A.G389A.K390A) showed better proliferation than the wild-type virus, while mutants 1–5 exhibited decreased in vitro infectivity, as determined by immunofluorescence assay (IFA). Based on a TMUV replicon system, the mutations exhibited no apparent effect on TMUV RNA replication. Subcellular fractionation assays and packaging system assays indicated that mutations in loops II–IV (T332A, T332S, S365A.S366A.T367A, and S388A.G389A.K390A, respectively) disrupted virion assembly. Moreover, loops I–IV played an important role in virus binding and entry, while mutant 6 (S388A.G389A.K390A) exhibited robust activity in virus entry. Taken together, our findings indicated the critical role of the loop regions in TMUV E domain III in the virus entry and assembly process. [ABSTRACT FROM AUTHOR]

Reported for the first time in 1955 in Malaysia, Tembusu virus (TMUV) remained, for a long time, in the shadow of flaviviruses with human health importance such as dengue virus or Japanese encephalitis virus. However, since 2010 and the first large epidemic in duck farms in China, the threat of its emergence on a large scale in Asia or even its spillover into the human population is becoming more and more significant. This review aims to report current knowledge on TMUV from viral particle organization to the development of specific vaccines and therapeutics, with a particular focus on host-virus interactions.

Ubiquitin-specific protease 18 (USP18) is known as an inhibition factor and has been associated with the innate immune response to pathogens. USP18 is the only deconjugating protease with specificity for interferon-stimulated gene 15 (ISG15), which is supposed to be missing in birds. To analyze the efficacy of goose USP18 (goUSP18) against Tembusu virus (TMUV) infection, we first cloned USP18 homologous cDNA from TMUV infected geese. The coding sequence was 1131 bp, and the deduced amino acid sequence shared conserved motifs with its homologues. Tissuespecific expression has shown that goUSP18 transcripts are strongly expressed in the spleen and liver of adult geese, as well as in the pancreas of goslings. Moreover, the goUSP18 transcripts were induced by goose interferons (goIFN) in goose embryo fibroblasts (GEF) and by TLR ligands in peripheral blood mononuclear cells (PBMC). Notably, goUSP18 transcripts were highly upregulated by TMUV infection compared to the basal level in uninfected birds. Taken together, these results suggested that goUSP18 was involved in host innate immunity against TMUV infection. [ABSTRACT FROM AUTHOR]

Flaviviruses pose a significant threat to public health worldwide. Recently, a novel flavivirus, duck Tembusu virus (TMUV), was identified as the causative agent of a serious duck viral disease in Asia. Its rapid spread and expanded host range have raised substantial concerns regarding its potential threat to non-avian hosts, including humans. However, the specific molecular host responses to this virus are poorly understood. In this study, we used the RNA-sequencing technique to analyse the differential gene expression in the spleens of infected goslings 5 days post-infection. In total, 2878 upregulated unigenes and 2943 downregulated unigenes were identified. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that different pattern recognition receptor (PRR) signalling pathways simultaneously participated in the sensing of the pathogen-associated molecular patterns (PAMPs) of TMUV, and the antigen presentation pathway and acquired immunity were activated. Then, the signals were transduced by the NF-kappa B (NF-κB) or the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways, resulting in the enormous production of various cytokines and interferon-stimulated genes (ISGs). We further investigated the immune response patterns in the liver and brain tissue using RT-qPCR. The bacterial peptidoglycan sensor nucleotide-binding oligomerization domain-containing protein 1 (NOD1) receptor was significantly upregulated, especially in the brain tissue, suggesting that NOD1 likely induces an inflammatory response by interacting with dsRNA, which is similar to its actions during hepatitis C viral (HCV) infection. However, major histocompatibility complex II (MHCII) was downregulated only in the spleen, indicating that the downregulation of MHCII in the spleen may be an immune evasion strategy of TMUV to facilitate pathogenesis during infection. Here, we are the first to report a transcriptome analysis of the host immune response to TMUV infection, and the data reported herein may help elucidate the molecular mechanisms of the gosling-TMUV interaction. [ABSTRACT FROM AUTHOR]

Since its outbreak in 2010, Tembusu virus (TMUV) has spread widely throughout China and Southeast Asia, causing significant economic losses to the poultry industry. In 2018, an attenuated vaccine called FX2010-180P (180P) was licensed for use in China. The 180P vaccine has demonstrated its immunogenicity and safety in mice and ducks. The potential use of 180P as a backbone for flavivirus vaccine development was explored by replacing the pre-membrane (prM) and envelope (E) genes of the 180P vaccine strain with those of Japanese encephalitis virus (JEV). Two chimeric viruses, 180P/JEV-prM-E and 180P/JEV-prM-E S156P with an additional E protein S156P mutation were successfully rescued and characterized. Growth kinetics studies showed that the two chimeric viruses replicated to similar titers as the parental 180P virus in cells. Animal studies also revealed that the virulence and neuroinvasiveness of the 180P/JEV-prM-E chimeric virus was decreased in mice inoculated intracerebrally (i.c.) and intranasally (i.n.), respectively, compared to the wild-type JEV strain. However, the chimeric 180P/JEV-prM-E virus was still more virulent than the parent 180P vaccine in mice. Additionally, the introduction of a single E S156P mutation in the chimeric virus 180P/JEV-prM-E S156P further attenuated the virus, which provided complete protection against challenge with a virulent JEV strain in the mouse model. These results indicated that the FX2010-180P could be used as a promising backbone for flavivirus vaccine development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xu, Liu, Yan, Teng, Yuan, Zhang, Li and Li.)

The Tembusu virus (TMUV) is an avian pathogenic flavivirus that causes a highly contagious disease and catastrophic losses to the poultry industry. The myxovirus resistance protein (Mx) of innate immune effectors is a key antiviral “workhorse” of the interferon (IFN) system. Although mammalian Mx resistance against myxovirus and retrovirus was witnessed for decades, whether or not bird Mx has anti-flavivirus activity remains unknown. In this study, we found that the transcription of goose Mx (goMx) was obviously driven by TMUV infection, both in vivo and in vitro, and that the titers and copies of TMUV were significantly reduced by goMx overexpression. In both primary (goose embryo fibroblasts, GEFs) and passaged cells (baby hamster kidney cells, BHK21, and human fetal kidney cells, HEK 293T), it was shown that goMx was mainly located in the cytoplasm, and sporadically distributed in the nucleus. The intracellular localization of this protein is attributed to the predicted bipartite nuclear localization signal (NLS; 30 residues: the 441st–471st amino acids of goMx). Intuitively, it seems that the cells with a higher level of goMx expression tend to have lower TMUV loads in the cytoplasm, as determined by an immunofluorescence assay. To further explore the antiviral determinants, a panel of variants was constructed. Two amino acids at the 125th (Lys) and 145th (Thr) positions in GTP-binding elements, not in the L4 loop (40 residues: the 532nd–572nd amino acids of goMx), were vital for the antiviral function of goMx against TMUV in vitro. These findings will contribute to our understanding of the functional significance of the antiviral system in aquatic birds, and the development of goMx could be a valuable therapeutic agent against TMUV.

Flavivirus RNA cap-methylation plays an important role in viral infection, proliferation, and escape from innate immunity. The methyltransferase (MTase) of the flavivirus NS5 protein catalyzes viral RNA methylation. The E218 amino acid of the NS5 protein MTase domain is one of the active sites of flavivirus methyltransferase. In flaviviruses, the E218A mutation abolished 2'-O methylation activity and significantly reduced N-7 methylation activity. Tembusu virus (TMUV , genus Flavivirus) was a pathogen that caused neurological symptoms in ducklings and decreased egg production in laying ducks. In this study, we focused on a comprehensive understanding of the effects of the E218A mutation on TMUV characteristics and the host immune response. E218A mutation reduced TMUV replication and proliferation, but did not affect viral adsorption and entry. Based on a TMUV replicon system, we found that the E218A mutation impaired viral translation. In addition, E218A mutant virus might be more readily recognized by RIG-I-like receptors to activate the corresponding antiviral immune signaling than WT virus. Together, our data suggest that the E218A mutation of TMUV MTase domain impairs viral replication and translation and may activates RIG-I-like receptor signaling, ultimately leading to a reduction in viral proliferation. [ABSTRACT FROM AUTHOR]

Tembusu virus ( TMUV) is a flavivirus, presumed to be a mosquito-borne flavivirus of the Ntaya virus subgroup. To date, however, there have been no reports indicating that mosquitoes are involved in the spread of TMUV. In this study, we report the first isolation of TMUV from Culex mosquitoes. We describe the isolation and characterization of a field strain of TMUV from mosquitoes collected in Shandong Province, China. The virus isolate, named TMUV- SDMS, grows well in mosquito cell line C6/36, in Vero and duck embryo fibroblast ( DEF) cell lines, and causes significant cytopathic effects in these cell cultures. The TMUV- SDMS genome is a single-stranded RNA, 10 989 nt in length, consisting of a single open reading frame encoding a polyprotein of 3410 amino acids, with 5′ and 3′ untranslated regions of 142 and 617 nt, respectively. Phylogenetic analysis of the E and NS5 genes revealed that the TMUV- SDMS is closely related to the TMUV YY5 and BYD strains which cause severe egg-drop in ducks. The 3′ NTR of TMUV- SDMS contains two pairs of tandem repeat CS and one non-duplicate CS, which have sequence similarities to the same repeats in the YY5 and BYD strains. Our findings indicate that mosquitoes carrying the TMUV may play an important role in the spread of this virus and in disease outbreak. [ABSTRACT FROM AUTHOR]

Unlike most flaviviruses transmitted by arthropods, Tembusu virus (TMUV) is still active during winter and causes outbreaks in some areas, indicating vector-independent spread of the virus. Gastrointestinal transmission might be one of the possible routes of vector-free transmission, which also means that the virus has to interact with more intestinal bacteria. Here, we found evidence that TMUV indeed can transmit through the digestive tract. Interestingly, using an established TMUV disease model by oral gavage combined with an antibiotic treatment, we revealed that a decrease in intestinal bacteria significantly reduced local TMUV proliferation in the intestine, revealing that the bacterial microbiome is important in TMUV infection. We found that lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria enhanced TMUV proliferation by promoting its attachment. Toll-like receptor 4 (TLR4), a cell surface receptor, can transmit signal from LPS. We confirmed colocalization of TLR4 with TMUV envelope (E) protein as well as their interaction in infected cells. Coherently, TMUV infection of susceptible cells was inhibited by an anti-TLR4 antibody, purified soluble TLR4 protein, and knockdown of TLR4 expression. LPS-enhanced TMUV proliferation could also be blocked by a TLR4 inhibitor. Meanwhile, pretreatment of duck primary cells with TMUV significantly impaired LPS-induced interleukin 6 production. Collectively, our study provides first insights into vector-free transmission mechanisms of flaviviruses., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Since 2010, Tembusu virus (TMUV) has spread widely in China, causing huge economic losses to the poultry industry. Due to the infectious and zoonotic nature of flaviviruses, their potential threat to public health is of great concern. Cellular immune responses usually play a critical role in combating viral infections. To study the molecular basis of cell immunity induced by TMUV, 14 cytotoxic T lymphocyte (CTL) epitope peptides of TMUV antigen E, NS1 and NS3 were predicted by bioinformatics tools. Their abilities to induce cellular immune responses were determined by IFN-γ ELISpot assay, and 4 peptides were found to exhibit highly significant responses upon stimulation. In addition, the cytotoxic activity induced by the epitope peptides was assessed by lactate dehydrogenase (LDH) release assay. Finally, among these peptides, we identified two murine TMUV NS3-derived H-2d-restricted CTL epitopes in BALB/c mice, which could be used to further study of epitope vaccines against TMUV infection. • Intracerebral inoculation with TMUV in mice can induce a specific cellular immune response. • Two novel TMUV NS3-derived H-2d-restricted CTL epitopes were identified in BALB/c mice. • The sequence of the peptide NS3 299–307 (GYISTKVEL) is highly conserved in flaviviruses. [ABSTRACT FROM AUTHOR]

Tembusu virus (TMUV), an avian mosquito-borne flavivirus, was first identified from Culex tritaeniorhynchus in 1955. To validate the effects of the 3'-untranslated region (3'UTR) in viral host-specific adaptation, we generated a set of chimeric viruses using CQW1 (duck strain) and MM 1775 (mosquito strain) as backbones with heterogeneous 3'UTRs. Compared with rMM 1775, rMM-CQ3'UTR (recombinant MM 1775 virus carrying the 3'UTR of CQW1) exhibited enhanced proliferation in vitro , with peak titers increasing by 5-fold in duck embryonic fibroblast (DEF) cells or 12-fold in baby hamster kidney (BHK-21) cells; however, the neurovirulence of rMM-CQ3'UTR was attenuated in 14-day-old Kunming mice via intracranial injection, with slower weight loss, lower mortality, and reduced viral loads. In contrast, rCQ-MM3'UTR showed similar growth kinetics in vitro and neurovirulence in mice compared with those of rCQW1. Then, the Stem-loop I (SLI) structure, which showed the highest variation within the 3'UTR between CQW1 and MM 1775, was further chosen for making chimeric viruses. The peak titers of rMM-CQ3'UTRSLI displayed a 15- or 4-fold increase in vitro , and the neurovirulence in mice was attenuated, compared with that of rMM 1775; rCQ-MM3'UTRSLI displayed comparable multiplication ability in vitro but was significantly attenuated in mice, in contrast with rCQW1. In conclusion, we demonstrated that the TMUV SLI structure of the 3'UTR was responsible for viral host-specific adaptation of the mosquito-derived strain in DEF and BHK-21 cells and regulated viral pathogenicity in 14-day-old mice, providing a new understanding of the functions of TMUV 3'UTR in viral host switching and the pathogenicity changes in mice. IMPORTANCE Mosquito-borne flaviviruses (MBFVs) constitute a large number of mosquito-transmitted viruses. The 3'-untranslated region (3'UTR) of MBFV has been suggested to be relevant to viral host-specific adaptation. However, the evolutionary strategies for host-specific fitness among MBFV are different, and the virulence-related structures within the 3'UTR are largely unknown. Here, using Tembusu virus (TMUV), an avian MBFV as models, we observed that the duck-derived SLI of the 3'UTR significantly enhanced the proliferation ability of mosquito-derived TMUV in baby hamster kidney (BHK-21) and duck embryonic fibroblast (DEF) cells, suggesting that the SLI structure was crucial for viral host-specific adaptation of mosquito-derived TMUVs in mammalian and avian cells. In addition, all SLI mutant viruses exhibited reduced viral pathogenicity in mice, indicating that SLI structure was a key factor for the pathogenicity in mice. This study provides a new insight into the functions of the MBFV 3'UTR in viral host switching and pathogenicity changes in mice.

The article focuses on a study related to the detection of Tembusu virus E gene from ducks by using loop-mediated isothermal amplification (LAMP) assay. Topics discussed include development of the assay using primers which targets the E gene of the virus, determination of the cross-reactivity with other viruses such as duck plagues virus, avian influenza virus, and Newcastle disease virus, and the significance of the LAMP assay in the molecular epidemiological investigation of the virus.

Reported for the first time in 1955 in Malaysia, Tembusu virus (TMUV) remained, for a long time, in the shadow of flaviviruses with human health importance such as dengue virus or Japanese encephalitis virus. However, since 2010 and the first large epidemic in duck farms in China, the threat of its emergence on a large scale in Asia or even its spillover into the human population is becoming more and more significant. This review aims to report current knowledge on TMUV from viral particle organization to the development of specific vaccines and therapeutics, with a particular focus on host-virus interactions. [ABSTRACT FROM AUTHOR]

• TMUV showed tropism in human liver, nerve and kidney cells. • The tropism suggests zoonotic risk and potential target organs of TMUV infection in humans. Tembusu virus (TMUV) causes disease in poultry, especially in ducks, resulting in abnormality in egg production and with high morbidity and mortality, resulting in great loss in duck farming industry in China and Southeast Asia. Previous studies on the pathogenesis of TMUV infection have been mostly conducted in poultry, with a few studies being undertaken in mice. While TMUV does not cause disease in humans, it has been reported that antibodies against TMUV have been found in serum samples from duck farmers, and thus data on TMUV infection in humans is limited, and the pathogenesis is unclear. In this study we investigated the cell tropism and potential susceptibility of humans to TMUV using several human cell lines. The results showed that human nerve and liver cell lines were both highly susceptible and permissive, while human kidney cells were susceptible and permissive, albeit to a lower degree. In addition, human muscle cells, lung epithelial cells, B-cells, T-cells and monocytic cells were largely refractory to TMUV infection. This data suggests that liver, neuron and kidney are potential target organs during TMUV infection in humans, consistent with what has been found in animal studies. [ABSTRACT FROM AUTHOR]

The CTL immune response mediated by MHC I plays an important role in duck anti-TMUV infection. This study reports the expression, purification and crystallization of a complex of duck MHC class I molecules Anpl-UAA*SD, duck β2-microglobulin (Anpl-β2m) and the polypeptide LRKRQLTVL (LRK9) derived from Tembusu virus (TMUV) NS3. The crystal diffraction resolution is 1.50 Å and belongs to the P6 2 space group, and the unit cell parameters are a = 82.468, b = 82.468, c = 112.507. The Matthew's constant is calculated to be 2.32 Å 3  Da -1 , and an asymmetric unit contains a complex molecule with a solvent content of 47%. The research lays the foundation for the structure of immune molecules about duck anti-TMUV research., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The Tembusu virus (TMUV) is an avian pathogenic flavivirus that causes a highly contagious disease and catastrophic losses to the poultry industry. The myxovirus resistance protein (Mx) of innate immune effectors is a key antiviral “workhorse” of the interferon (IFN) system. Although mammalian Mx resistance against myxovirus and retrovirus was witnessed for decades, whether or not bird Mx has anti-flavivirus activity remains unknown. In this study, we found that the transcription of goose Mx (goMx) was obviously driven by TMUV infection, both in vivo and in vitro, and that the titers and copies of TMUV were significantly reduced by goMx overexpression. In both primary (goose embryo fibroblasts, GEFs) and passaged cells (baby hamster kidney cells, BHK21, and human fetal kidney cells, HEK 293T), it was shown that goMx was mainly located in the cytoplasm, and sporadically distributed in the nucleus. The intracellular localization of this protein is attributed to the predicted bipartite nuclear localization signal (NLS; 30 residues: the 441st–471st amino acids of goMx). Intuitively, it seems that the cells with a higher level of goMx expression tend to have lower TMUV loads in the cytoplasm, as determined by an immunofluorescence assay. To further explore the antiviral determinants, a panel of variants was constructed. Two amino acids at the 125th (Lys) and 145th (Thr) positions in GTP-binding elements, not in the L4 loop (40 residues: the 532nd–572nd amino acids of goMx), were vital for the antiviral function of goMx against TMUV in vitro. These findings will contribute to our understanding of the functional significance of the antiviral system in aquatic birds, and the development of goMx could be a valuable therapeutic agent against TMUV. [ABSTRACT FROM AUTHOR]