Tembusu Virus Research Articles

PUC18-CpG Is an Effective Adjuvant for a Duck Tembusu Virus Inactivated Vaccine.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus responsible for massive economic losses in the duck industry. However, commercially inactivated DTMUV vaccines have been ineffective at inducing protective immunity in ducks. The widely used adjuvant cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) reportedly improve humoral and cellular immunities in animal models. However, its effectiveness in DTMUV vaccines requires validation. Here, we assessed the protective efficacy of pUC18-CpG as an adjuvant in an inactivated live DTMUV vaccine in ducks. Our results revealed that the serum hemagglutination inhibition (HI) antibody titers, positive rates of anti-DTMUV antibodies, the concentration of serum cytokines, and protection efficacy were significantly increased in ducks immunized with pUC18-CpG compared to that in the control group. Moreover, ducks immunized with a full vaccine dose containing a half dose of antigen supplemented with 40 μg of pUC18-CpG exhibited the most potent responses. This study suggests that pUC18-CpG is a promising adjuvant against DTMUV, which might prove effective in treating other viral diseases in waterfowl.

High-mobility group box 1 protein (HMGB1) from Cherry Valley duck mediates signaling pathways and antiviral activity

High-mobility group box 1 protein (HMGB1) shows endogenous damage-associated molecular patterns (DAMPs) and is also an early warning protein that activates the body’s innate immune system. Here, the full-length coding sequence of HMGB1 was cloned from the spleen of Cherry Valley duck and analyzed. We find that duck HMGB1(duHMGB1) is mostly located in the nucleus of duck embryo fibroblast (DEF) cells under normal conditions but released into the cytoplasm after lipopolysaccharide (LPS) stimulation. Knocking-down or overexpressing duHMGB1 had no effect on the baseline apoptosis rate of DEF cells. However, overexpression increased weakly apoptosis after LPS activation. In addition, overexpression strongly activated the IFN-I/IRF7 signaling pathway in DEF cells and significantly increased the transcriptional level of numerous pattern recognition receptors (PRRs), pro-inflammatory cytokines (IL-6, TNF-α), IFNs and antiviral molecules (OAS, PKR, Mx) starting from 48 h post-transfection. Overexpression of duHMGB1 strongly impacted duck virus replication, either by inhibiting it from the first stage of infection for novel duck reovirus (NDRV) and at late stage for duck Tembusu virus (DTMUV) or duck plague virus (DPV), or promoting replication at early stage for DTMUV and DPV infection. Importantly, data from duHMGB1 overexpression and knockdown experiments, time-dependent DEF cells transcriptional immune responses suggest that duHMGB1 and RIG-I receptor might cooperate to promote the expression of antiviral proteins after NDRV infection, as a potential mechanism of duHMGB1-mediated antiviral activity.

Duck Tembusu virus detection and characterization from mosquitoes in duck farms, Thailand.

Duck Tembusu virus (DTMUV), an emerging infectious disease in ducks, belongs to the Flavivirus genus and Flaviviridae family. The transmission of DTUMV involves mosquito vectors; however, the exact role of mosquitoes in the ecology of DTMUV in Thailand remains unclear. This study was conducted to examine DTMUV detection and characterization from mosquitoes in duck farms in central Thailand. Mosquitoes were collected from two duck farms in Sing Buri Province and two duck farms in Ang Thong Province from September 2015 to July 2016 using four CDC-light traps. A total of 30,841 mosquitoes were collected and identified to seven species (Anopheles (An.) barbirostris, An.stephensi, Culex (Cx.) gelidus, Cx.quinquefasciatus, Cx.tritaeniorhynchus, Mansonia (Ma.) annulifera and Ma.uniformis). The most common collected species from each duck farm and each collection time was Cx.tritaeniorhynchus. Mosquitoes were pooled according to species, location, and collection time and then examined for DTMUV by RT-PCR. A total of 273 mosquito pools were examined, with only one pool of Cx.tritaeniorhynchus collected from Sing Buri Province in November 2015 testing positive for DTMUV. Phylogenetic analysis of the polyprotein genes demonstrated that a mosquito-derived Thai DTMUV was grouped into subcluster 2.1 and most closely related to the 2013 Thai DTMUVs. Thus, this study indicated that Cx.tritaeniorhynchus may play a role as a vector in the transmission of DTMUV in Thailand. However, additional studies concerning the vector competence of this mosquito for DTMUV are needed.

DEF Cell-Derived Exosomal miR-148a-5p Promotes DTMUV Replication by Negative Regulating TLR3 Expression.

Duck tembusu virus (DTMUV) is a single-stranded, positive-polarity RNA flavivirus that has caused considerable economic losses in China in recent years. Innate immunity represents the first line of defense against invading pathogens and serves as an important role in resisting viral infections. In this study, we found that the infection of ducks by DTMUV triggers Toll-like receptors (TLRs) and (RIG-I)-like receptors (RLRs) signaling pathways and inducing abundant of pro-inflammatory factors and type I interferons (IFNs), in which melanoma differentiation-associated gene 5 (MDA5) and Toll-like receptor 3 (TLR3) play important immunity roles, they can inhibit the replication process of DTMUV via inducing type I IFNs. Moreover, we demonstrated that type I IFNs can inhibit the DTMUV replication process in a time- and dose-dependent manner. Exosomes are small membrane vesicles that have important roles in intercellular communication. MicroRNAs (miRNAs) are small non-coding RNAs that can modulate gene expression and are common substances in exosomes. In our experiment, we successfully isolated DEF cells derived exosome for the first time and explored its function. Firstly, we found the expression of miR-148a-5p is significantly decreased following DTMUV infect. Then we found miR-148a-5p can target TLR3 and down-regulate the expression of TLR3, serving as a negative factor in innate immunity. Unfortunately, we cannot find miRNAs with different expression changes that can target MDA5. Lastly, our experimental results showed that TLR3 was one of the causes of miR-148a-5p reduction, suggesting that the high level of TLR3 after DTMUV infect can both trigger innate immunity and suppress miR-148a-5p to resist DTMUV.

Autophagy Promotes Duck Tembusu Virus Replication by Suppressing p62/SQSTM1-Mediated Innate Immune Responses In Vitro.

Duck Tembusu virus (DTMUV) has recently appeared in ducks in China and the key cellular determiners for DTMUV replication in host cells remain unknown. Autophagy is an evolutionarily conserved cellular process that has been reported to facilitate flavivirus replication. In this study, we utilized primary duck embryo fibroblast (DEF) as the cell model and found that DTMUV infection triggered LC3-II increase and polyubiquitin-binding protein sequestosome 1 (p62) decrease, confirming that complete autophagy occurred in DEF cells. The induction of autophagy by pharmacological treatment increased DTMUV replication in DEF cells, whereas the inhibition of autophagy with pharmacological treatments or RNA interference decreased DTMUV replication. Inhibiting autophagy enhanced the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and interferon regulatory factor 7 (IRF7) pathways and increased the p62 protein level in DTMUV-infected cells. We further found that the overexpression of p62 decreased DTMUV replication and inhibited the activation of the NF-κB and IRF7 pathways, and changes in the NF-κB and IRF7 pathways were consistent with the level of phosphorylated TANK-binding kinase 1 (p-TBK1). Opposite results were found in p62 knockdown cells. In summary, we found that autophagy-mediated p62 degradation acted as a new strategy for DTMUV to evade host innate immunity.

The prokaryotic expression, polyclonal antibody preparation, and subcellular localization of the transmembrane protein NS2A of the duck Tembusu virus

Duck Tembusu virus (DTMUV) is a single-stranded, positive-sense RNA arbovirus, belonging to the genus Flavivirus, the family Flaviviridae. As a transmembrane protein, non-structural protein 2A (NS2A) plays an important role in virion assembly, replication complex and antagonizing host immune response. Since NS2A protein contains many hydrophobicaminoacids, it is hard to gain the full-length protein of NS2A for prokaryotic expression. Therefore, to make a deep study, prokaryotic expression and polyclonal antibody preparation of truncated DTMUV NS2A was performed. The truncated NS2A gene (178-450 bp) was obtained, and sub-cloned into the prokaryotic vector pGEX-4T-1 (pGEX-4T-1-NS2A178-450bp). Subsequently, the recombinant GST-NS2A60-150aa protein was successfully expressed in E. coli BL21 (DE3) with the induction by 0.3 mmol/l isopropyl β-D-thiogalactoside (IPTG) for 6 h at 37°C. The GST-NS2A60-150aa protein was extracted from the gel. The BALB/c mice were immunized with the purified recombinant NS2A protein to prepare polyclonal antibodies against the truncated NS2A protein. The titer of the polyclonal antibodies, determined by ELISA analysis, was 1:128,000. The specificity of the polyclonal antibodies (mPAb-DTMUV-NS2A60-150aa) were verified by Western blot analysis. Furthermore, the indirect immunofluorescence (IFA) was performed to explore the subcellular localization of NS2A. NS2A protein was, in the transfected cells, located mainly around nucleus in the endoplasmatic reticulum. Taken together, our study provided a useful tool for the further exploration of the biological functions and molecular mechanism of DTMUV NS2A. Keywords: duck Tembusu virus; non-structural protein 2A; prokaryotic expression; polyclonal antibodies; subcellular location.

Molecular identification of duck DDX3X and its potential role in response to Tembusu virus

ATP-dependent DEAD (Asp-Glu-Ala-Asp)-box RNA helicases not only regulate RNA metabolism, but also are involved in host antiviral innate immune responses. It is important to investigate the orthologs of this protein family to broaden our understanding of innate immunity and promote protective strategies against viral infections in ducks. In the current study, duck DDX3X (duDDX3X) was first cloned, which consists of 1959 bp encoding a protein of 652 amino acids. duDDX3X has the typical structure of this family, including nine motifs, DEAD and HELICc domains. The amino acid sequence of duDDX3X shares a high similarity with the DDX3Xs of avian and mammalian. Quantitative real-time PCR indicated that duDDX3X was ubiquitously expressed in nearly all tissues. Overexpression of duDDX3X could activate interferon (IFN)-β and enhance the RIG-I-induced IFN-β yield in duck embryo fibroblast cells. However, duDDX3X had no significant effect on the expression of proinflammatory cytokines such as IL-1β, IL-6, and CXCL-8. Tembusu virus (TMUV) infection significantly downregulated duDDX3X. Overexpression and siRNA interference studies showed that duDDX3X inhibited the replication of TMUV through IFN-β at the early stages of infection. Collectively, our results indicated that duDDX3X could positively modulate type I interferon and play an essential role in response to TMUV infection. This study will contribute to a better understanding of duDDX3X in the innate immune system of ducks and lay a solid foundation for further studies of duDDX3X in antiviral immunity.

Development and application of an indirect ELISA for the serological detection of duck Tembusu virus infection based on the NS1 protein antigen.

Duck Tembusu virus (DTMUV) has caused significant economic losses in China since 2010. However, there is still a lack of effective methods to diagnose the disease caused by this virus, and especially to differentiate infection from vaccination. In this study, we established a novel indirect enzyme-linked immunosorbent assay (iELISA) and performed a retrospective serological survey for DTMUV in Anhui province, China. Our results show that the iELISA displayed high specificity sensitivity, and with no serological cross-reaction with other duck pathogens. These findings indicate that the newly developed iELISA could be a useful screening tool for large-scale monitoring of the epidemiology of DTMUV infection in ducks.

Binding of the Duck Tembusu Virus Protease to STING Is Mediated by NS2B and Is Crucial for STING Cleavage and for Impaired Induction of IFN-β.

Duck Tembusu virus (DTMUV) is a newly emerged causative agent of avian disease. The protease-dependent immune evasion of flaviviruses has been reported; however, the molecular details of this process are unclear. In this study, we found that DTMUV nonstructural protein 2B-3, a NS2B3 protease, can inhibit IFN-β production. DTMUV NS2B3 inhibited RIG-I-, MDA5-, MAVS-, and STING-directed IFN-β transcription, but not TBK1- and IRF7-mediated induction of IFN-β. Further analysis showed that DTMUV NS2B3 could cleave duck STING (duSTING); the cleavage was dependent on the protease activity of NS2B3. Moreover, the STING cleavage event occurred in a not-strictly-species-specific manner. The scissile bond of duSTING cleaved by NS2B3 was mapped between the R84 and G85 residues. The ability of NS2B3 to reduce duSTING cleavage-resistant mutant-mediated IFN-β, and ISG production was significantly reduced, demonstrating that duSTING cleavage is essential for NS2B3-induced suppression of type I IFN responses. Remarkably, the binding of NS2B3 to duSTING, which is a prerequisite for cleavage, was found to depend on NS2B, but not NS3, the cofactor of the enzyme. Unexpectedly, we found that the region between aa residues 221-225 of duSTING, distal from the site of the scissile bond, was essential for the binding of NS2B3 to duSTING and/or the cleavage of duSTING by NS2B3. Thus, we identified the molecular mechanism by which DTMUV subverts the host innate immunity using its protease. More importantly, our study provides insight into NS2B3-mediated STING cleavage events in general.

Detection of Neutralizing Antibodies to Tembusu Virus: Implications for Infection and Immunity.

Neutralizing antibodies are the key mediators of protective immune response to flaviviruses after both infection and vaccination. Plaque reduction neutralization test (PRNT) is considered the “gold standard” for measurement of the immunity. To date, little is known regarding neutralizing antibody response to Tembusu virus (TMUV), a novel flavivirus emerging in ducks in 2010. Here, we developed a PRNT for detection of TMUV neutralizing antibodies. Following optimization and validation, the PRNT was applied to test serum samples from different flocks of ducks. Using sera prepared in experimental conditions, the levels of 50% end point titer (neutralizing dose, ND50) generated from positive sera (5,012–79,433) were significantly higher than those from mock-infected sera (10 to 126), indicating that the test can be used in the detection of TMUV-specific neutralizing antibodies. Dose-dependent efficacy test of a cell-derived 180th passage of a plaque-purified virus of the PS TMUV isolate (PS180) in combined with immunization-challenge experiments revealed that ND50 titer of ~1,258 is the minimum capable of providing adequate protection against challenge with virulent TMUV. In the investigation of serum samples collected from three flocks infected by TMUV and four flocks vaccinated with a licensed attenuated vaccine (the 120th passage virus), ND50 titers peaked at 1 week after both disease onset (7,943–125,893) and vaccination (3,612–79,432), and high levels of ND50 titer were detected in sera collected at 15 weeks after disease onset (5,012–63,095) and 17 weeks after vaccination (3,981–25,119). Together these findings demonstrated that spontaneous and experimental infections by TMUV and vaccination with the licensed TMUV attenuated vaccine elicit high, long-lasting neutralizing antibodies. The highest ND50 titer of neutralizing antibodies elicited by PS180 was determined to be 3,162, suggesting that attenuation of TMUV by more passages has a dramatic impact on the neutralizing antibody response of the virus.

Avian Flavivirus Enters BHK-21 Cells by a Low pH-Dependent Endosomal Pathway.

Duck Tembusu virus (DTMUV), a pathogenic member of the Flavivirus family, was first discovered in the coastal provinces of South-Eastern China in 2010. Many previous reports have clearly shown that some Flaviviruses utilize several endocytic pathways to enter the host cells, however, the detailed mechanism of DTMUV entry into BHK-21 cells, which is usually employed to produce commercial veterinary vaccines for DTMUV, as well as of other Flaviviruses by serial passages, is still unknown. In this study, DTMUV entry into BHK-21 cells was found to be inhibited by noncytotoxic concentrations of the agents chloroquine, NH4Cl, and Bafilomycin A1, which blocked the acidification of the endosomes. Inactivation of virions by acid pretreatment is a hallmark of viruses that utilize a low-pH-mediated entry pathway. Exposure of DTMUV virions to pH 5.0 in the absence of host cell membranes decreased entry into cells by 65%. Furthermore, DTMUV infection was significantly decreased by chlorpromazine treatment, or by knockdown of the clathrin heavy chain (CHC) through RNA interference, which suggested that DTMUV entry depends on clathrin. Taken together, these findings highlight that a low endosomal pH is an important route of entry for DTMUV.

Characterization and genomic analysis of emerging astroviruses causing fatal gout in goslings.

Since February 2017, severe outbreaks of fatal gout caused by novel gosling astroviruses (GoAstVs) have occurred in several Chinese provinces, causing a considerable economic impact on the poultry industry. To assess the infection status of GoAstVs causing gout, 165 clinical samples were collected from goslings from seven farms located in different Chinese provinces, and they were screened for viral infection. Seven GoAstV strains were completely sequenced. The positive infection rates of GoAstV, goose parvovirus, reovirus, goose haemorrhagic polyomavirus and Tembusu virus were 100%, 9.69%, 3.64%, 0% and 0%, respectively, indicating the role of GoAstV in gout. The genomes of all seven GoAstV strains were 7170-nt long and encoded three open reading frames (ORFs), namely, ORF1a, ORF1b and ORF2. Sequence and phylogenetic analyses of the seven GoAstV strains showed that these were avastroviruses and were closely related to viruses classified within Avastrovirus 3 and turkey astrovirus 2. Moreover, the mutation rates of ORF1a and ORF2 were high, and ORF1a was highly mutated at amino acid loci 545-580. The tertiary structure of the mutated ORF2 protein was smooth, and its antigenic epitope was highly mutated, which may be related to the pathogenicity of the virus and caused by antibody pressure from the host. These findings enrich our understanding of the evolution of novel GoAstVs causing gout and their circulation as well as lay the foundation for the selection of vaccine strains.

Molecular cloning and functional characterization of duck Janus kinase 1

Janus kinase 1 (JAK1) is a member of JAK family of non-receptor protein tyrosine kinases that plays critical roles in transducing cytokine signals via JAK-signal transducer and activator of transcription (STAT) signaling pathway. The importance of JAK1 in innate immunity has been well-studied in mammals and fish, yet in avian remains largely unknown. Here, we cloned the full-length of the duck JAK1 (duJAK1) gene for the first time. DuJAK1 encoded a protein of 1152 amino acids and possessed high amino acid identity with goose and budgerigar JAK1s. The duJAK1 was expressed in all detected tissues, especially high in the thymus and bursa of Fabricius. Overexpression of duJAK1 significantly activated ISRE promoter activity and induced duck viperin, 2′, 5′-OAS, MX, PKR and ZAP expression. Knockdown of duJAK1 by small interfering RNA significantly inhibited duck Tembusu virus (DTMUV)-, duck Enteritis virus (DEV)-, poly (I:C)-, poly (dA:dT)- or Sendai virus (SeV)-induced ISRE promoter activation. Furthermore, duJAK1 exhibited antiviral activity against DTMUV infection. These results will help us understand the function of JAK family proteins in duck antiviral immunity.

The truncated E protein of DTMUV provide protection in young ducks

Duck Tembusu virus (DTMUV) is a major pathogen of duck industry in China. In the current study, we generated different constructs containing envelope (E) protein, pre-membrane-envelope (prM-E) protein, and C-terminally truncated E protein of the DTMUV. The constructed proteins could induce specific antibody responses in young ducks. When ducklings were immunized with the constructed proteins, they were 100% protected against DTMUV infection. Furthermore, the fluorescent signal of the truncated E protein was stronger than other constructed proteins, when Bac-to-Bac baculovirus expression system was applied. Our data demonstrated that the truncated E protein used in the current study could be applied as a potential vaccine candidate to control DTMUV infection in young ducks.

Heparin sulfate is the attachment factor of duck Tembus virus on both BHK21 and DEF cells

BackgroundDuck tembusu virus (DTMUV, genus Flaviviruses, family Flaviviridae) is an emerging flavivirus that can infect a wide range of cells and cell lines in vitro, though the initial step of virus invasion remains obscure.MethodsIn this study, drug treatments that including heparin, chondroitin sulfate, heparinase I, chondroitinase ABC and trypsin were applied to detect the influence of DTMUV absorption, subsequently, the copy number of viral genome RNA was analyzed by quantitative real-time PCR. The inhibition process of viral absorption or entry by heparin was determined by western blotting, and the cytotoxicity of drug treated cells was detected by cell counting kit-8.ResultsWe found that the desulfation of glycosaminoglycans (GAGs) with sodium chlorate had a significant effect on the adsorption of DTMUV in both BHK21 and DEF cells. Based on this result, we incubated cells with a mixture of DTMUV and GAGs competition inhibitors or pre-treated cells with inhibitors, after incubation with the virus, the NS5 expression of DTMUV and viral titers were detected. The data suggested that heparin can significantly inhibit the absorption of DTMUV in a dose dependent manner but not at the step of viral entry in BHK21 and DEF cells. Meanwhile, heparinase I can significantly inhibit DTMUV attachment step.ConclusionsOur results clearly proved that heparin sulfate plays an important role in the first step of DTMUV entry, viral attachment, in both BHK21 and DEF cells, which sheds light on the entry mechanism of DTMUV.

Long-term passage of duck Tembusu virus in BHK-21 cells generates a completely attenuated and immunogenic population with increased genetic diversity

Duck Tembusu virus (TMUV) is an emerging pathogenic flavivirus that causes severe egg-drop and fatal encephalitis in domestic ducks and geese. Although a live-attenuated virus vaccine is effective for disease control, the stability of the attenuation has not been clearly evaluated due to a poor understanding of the attenuation mechanism. Here, a virulent duck TMUV isolate was successively passaged in BHK-21 cells, leading to an approximately 100-fold increase of virus production in cell culture and a complete attenuation of virulence for ducks. The passaged virus induced high titers of TMUV-specific antibody and provided efficient protection against a virulent TMUV challenge after a single-dose vaccination. One hundred and two, and eighteen single-nucleotide polymorphisms (SNPs) at a frequency of >1% were respectively identified in the attenuated virus population and the original isolate by deep sequencing. The increased SNPs numbers suggested that the accumulated variants of virus population may have conferred the phenotypic changes. We cloned and characterized a dominant variant exhibiting similar fitness to the mixed population, and 23 amino acid substitutions were identified across the viral open reading frame. Using reverse genetics, two chimeric viruses were generated by introducing the mutated E or NS5 gene into the backbone of virulent TMUV. We found that mutations in the E gene conferred a fitness advantage in BHK-21 cells and decreased the virus pathogenicity, whereas NS5 mutations reduced the virus infectivity in ducklings without altering the in vitro fitness. In conclusion, increased mutations in a virulent TMUV strain did substantially reduce the virus virulence, and mutations in multiple genes co-contribute to TMUV attenuation.

Avian Flavivirus Infection of Monocytes/Macrophages by Extensive Subversion of Host Antiviral Innate Immune Responses.

Avian Tembusu virus (TMUV) is a newly emerging avian pathogenic flavivirus in China and Southeast Asia with features of rapid spread, an expanding host range, and cross-species transmission. The mechanisms of its infection and pathogenesis remain largely unclear. Here, we investigated the tropism of this arbovirus in peripheral blood mononuclear cells of specific-pathogen-free (SPF) ducks and SPF chickens and identified monocytes/macrophages as the key targets of TMUV infection. In vivo studies in SPF ducks and SPF chickens with monocyte/macrophage clearance demonstrated that the infection of monocytes/macrophages was crucial for viral replication, transmission, and pathogenesis. Further genome-wide transcriptome analyses of TMUV-infected chicken macrophages revealed that host antiviral innate immune barriers were the major targets of TMUV in macrophages. Despite the activation of major pattern recognition receptor signaling, the inductions of alpha interferon (IFN-α) and IFN-β were blocked by TMUV infection on transcription and translation levels, respectively. Meanwhile, TMUV inhibited host redox responses by repressing the transcription of genes encoding NADPH oxidase subunits and promoting Nrf2-mediated antioxidant responses. The recovery of either of the above-mentioned innate immune barriers was sufficient to suppress TMUV infection. Collectively, we identify an essential step of TMUV infection and reveal extensive subversion of host antiviral innate immune responses.IMPORTANCE Mosquito-borne flaviviruses include a group of pathogenic viruses that cause serious diseases in humans and animals, including dengue, West Nile, and Japanese encephalitis viruses. These flaviviruses are zoonotic and use animals, including birds, as amplifying and reservoir hosts. Avian Tembusu virus (TMUV) is an emerging mosquito-borne flavivirus that is pathogenic for many avian species and can infect cells derived from mammals and humans in vitro Although not currently pathogenic for primates, the infection of duck industry workers and the potential risk of TMUV infection in immunocompromised individuals have been highlighted. Thus, the prevention of TMUV in flocks is important for both avian and mammalian health. Our study reveals the escape of TMUV from the first line of the host defense system in the arthropod-borne transmission route of arboviruses, possibly helping to extend our understanding of flavivirus infection in birds and refine the design of anti-TMUV therapeutics.

Pathogenicity of egg-type duck-origin isolate of Tembusu virus in Pekin ducklings

BackgroundTembusu virus (TMUV) usually affects adult ducks, causing a severe drop of egg production. It has also been shown to be pathogenic in commercial Pekin ducklings below 7 weeks of age. Here, we report a TMUV-caused neurological disease in young egg-type ducklings and the pathogenicity of the egg-type duck-origin TMUV isolates in meat-type Pekin ducklings.ResultsThe disease occurred in 25 to 40-day-old Jinding ducklings in China, and was characterized by paralysis. Gross lesions were lacking and microscopic lesions appeared chiefly in brain and spleen. Inoculation in embryonated duck eggs resulted in isolation of TMUV Y and GL. The clinical signs and microscopic lesions observed in the spontaneously infected egg-type ducks were repeated in Pekin ducklings by experimental infection. Notably, both Y and GL strains caused 100% mortality in the case of 2-day-old inoculation by intracerebral route. High mortalities (80 and 70%) also occurred following infection of the Y virus at 2 days of age by intramuscular route and at 9 days of age by intracerebral route.ConclusionsThese findings demonstrate that the egg-type duck-origin TMUVs exhibit high pathogenicity in Pekin ducklings, and that the severity of the disease in ducklings is dependent on the infection route and the age of birds at the time of infection. The availability of the highly pathogenic TMUV strains provides a useful material with which to begin investigations into the molecular basis of TMUV pathogenicity in ducks.

Evaluation of immunogenicity and protective efficacy of a CpG-adjuvanted DNA vaccine against Tembusu virus

Tembusu virus (TMUV) is a contagious pathogen of waterfowl including ducks and geese, which causes symptoms of high fever, loss of appetite and reduced egg production. The development of an effective vaccine is important for the prevention and control of the disease. We evaluated a DNA vaccine based on a recombinant pre-membrane (prM) and envelope (E) protein, using CpG oligodeoxynucleotide (ODN) as an adjuvanted, and tested it for protection efficacy. BHK21 cells were transfected with the recombinant plasmid pVAX1-prM/E-CpG, and the antigenicity of the expressed protein was detected using an indirect immunofluorescence assay (IFA) and western blot assay. One-day-old ducklings were intramuscularly injected with 200 μg doses of pVAX1-prM/E-CpG or pVAX1-CpG, or PBS at ten day intervals. The neutralizing antibodies and cell-mediated immune responses elicited by the DNA vaccine were detected using serum neutralization tests (SNTs) and ELISAs. At 20 days old, the ducks were challenged with 103EID50 doses of TMUV SD/02 strain and observed for 15 days post challenge. After the second DNA vaccination and during the monitoring period, the levels of TMUV neutralizing antibodies increased in the pVAX1-prM/E-CpG vaccinated ducks. Vaccination with pVAX1-prM/E-CpG resulted in 100.0% protection of the ducks, whereas approximately 40% of ducks vaccinated with pVAX-CpG or PBS manifested clinical symptoms. Expressions of IFN-γ and IL-6 in the pVAX1-prM/E-CpG group were significantly increased (p < 0.01) compared with the control groups during the entire experimental period. The results revealed that a vaccine co-expressing prM and E, and using a CpG-ODN motif as an adjuvant, could elicit effective neutralizing antibody titers and provide efficient protection to ducks against TMUV infection.

Development of chimeric virus-like particles containing the E glycoprotein of duck Tembusu virus

Duck Tembusu virus (DTMUV) has caused enormous economic losses to the poultry industry in China. In the current study, we generated chimeric virus-like particles (VLPs) containing E protein of the DTMUV and HA2 protein of the H3N2 avian influenza virus (AIV). The chimeric VLPs could induce specific antibody responses in both mice (n = 5/group) and ducks (n = 10/group). After immunizing ducklings with the chimeric VLPs, all immunized ducks (n = 10/group) were 100% (10/10) protected against homologous DTMUV strain and virus shedding was not detected on day 5 post-challenge, whereas 60% (6/10) of the ducklings immunized with PBS presented typical symptoms with a virus shedding rate of 90% (9/10). Furthermore, viral loads were significantly decreased in the birds of the chimeric VLPs immunized group, comparing to that of the PBS immunized group. Our data demonstrated that the chimeric VLPs used in the current study could be applied as a potential vaccine candidate to control DTMUV infections in young ducks.