Duck Tembusu Virus Infection Research Articles

Efficacy Evaluation of an Inactivated Duck Tembusu Virus Vaccine.

To evaluate the potential use of an inactivated virus-based vaccine for the control and prevention of the newly emerged duck Tembusu virus infection in China, a duck Tembusu virus isolate, Tembusu-HB, was propagated in 12-day-old duck embryos and inactivated by treatment with formaldehyde. The inactivated viral antigen was emulsified with mineral oil, and five batches of the vaccine were manufactured. The immunogenicity and protection efficacy of the vaccine were evaluated in Beijing ducks and Beijing white geese. Results showed that more than 80% of immunized ducks were protected against virulent virus challenge after two intramuscular or subcutaneous injections of the inactivated vaccine, as evidenced by the negative virus isolation results. The protection is also correlated with a positive virus-specific antibody response as detected by ELISA. In contrast, none of the control ducks and geese had any detectable antibody response. Virus was isolated from all control ducks and geese after virulent virus challenge. Interestingly, a variable level of protection (20%-80%) was observed in Beijing white geese immunized twice with the same batches of vaccine, suggesting a species-specific effect of the vaccine. Overall, the results clearly suggest that the inactivated duck Tembusu virus vaccine is immunogenic and provides protection against virulent virus challenge.

Immune responses of ducks infected with duck Tembusu virus.

Duck Tembusu virus (DTMUV) can cause serious disease in ducks, characterized by reduced egg production. Although the virus has been isolated and detection methods developed, the host immune responses to DTMUV infection are unclear. Therefore, we systematically examined the expression of immune-related genes and the viral distribution in DTMUV-infected ducks, using quantitative real-time PCR. Our results show that DTMUV replicates quickly in many tissues early in infection, with the highest viral titers in the spleen 1 day after infection. Rig-1, Mda5, and Tlr3 are involved in the host immune response to DTMUV, and the expression of proinflammatory cytokines (Il-1β, –2, –6, Cxcl8) and antiviral proteins (Mx, Oas, etc.) are also upregulated early in infection. The expression of Il-6 increased most significantly in the tissues tested. The upregulation of Mhc-I was observed in the brain and spleen, but the expression of Mhc-II was upregulated in the brain and downregulated in the spleen. The expression of the interferons was also upregulated to different degrees in the spleen but that of the brain was various. Our study suggests that DTMUV replicates rapidly in various tissues and that the host immune responses are activated early in infection. However, the overexpression of cytokines may damage the host. These results extend our understanding of the immune responses of ducks to DTMUV infection, and provide insight into the pathogenesis of DTMUV attributable to host factors.

Characterization of monoclonal antibodies against duck Tembusu virus E protein: an antigen-capture ELISA for the detection of Tembusu virus infection

The E protein of flaviviruses is the primary antigen that induces protective immunity, but a monoclonal antibody (mAb) against the E protein of duck Tembusu virus (DTMUV) has never been characterized. Six hybridoma cell lines secreting DTMUV anti-E mAbs were prepared and designated 2A5, 1F3, 1G2, 1B11, 3B6, and 4F9, respectively. An immunofluorescence assay indicated that the mAbs could specifically bind to duck embryo fibroblast (DEF) cells infected with DTMUV and that the E protein was distributed in the cytoplasm of the infected cells. Immunoglobulin isotyping differentiated the mAbs as IgG1 (1G2, 1B11, 4F9, 1F3, and 2A5) and IgG2b (3B6). The mAbs were used to identify three epitopes, A (2A5, 1F3, and 1G2), B (1B11 and 4F9), and C (3B6) on the E protein on the basis of a competitive binding assay. By using mAbs 1F3 and 3B6, we developed an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect E antigen from clinical samples. The AC-ELISA did not react with other known pathogens, indicating that the mAbs are specific for DTMUV. Compared to RT-PCR, the specificity and sensitivity of the AC-ELISA was 94.1 % and 98.0 %, respectively. This AC-ELISA thus represents a sensitive and rapid method for detecting DTMUV infection in birds.

The vaccine efficacy of recombinant duck enteritis virus expressing secreted E with or without PrM proteins of duck tembusu virus

A newly emerged tembusu virus that causes egg-drop has been affecting ducks in China since 2010. Currently, no vaccine is available for this disease. A live attenuated duck enteritis virus (DEV; a herpesvirus) vaccine has been used routinely to control lethal DEV in ducks since the 1960s. Here, we constructed two recombinant DEVs by transfecting overlapping fosmid DNAs. One virus, rDEV-TE, expresses the truncated form of the envelope glycoprotein (TE) of duck tembusu virus (DTMUV), and the other virus, rDEV-PrM/TE, expresses both the TE and pre-membrane proteins (PrM). Animal study demonstrated that both recombinant viruses induced measurable anti-DTMUV neutralizing antibodies in ducks. After two doses of recombinant virus, rDEV-PrM/TE completely protected ducks from DTMUV challenge, whereas rDEV-TE only conferred partial protection. These results demonstrate that recombinant DEV expressing the TE and pre-membrane proteins is protective and can serve as a potential candidate vaccine to prevent DTMUV infection in ducks.

Tembusu virus infection in Cherry Valley ducks: The effect of age at infection

Three groups of Cherry Valley ducks at 5 day, 2 week and 5 week of age were intranasally infected with the WFCL strain of Tembusu virus (TMUV) to investigate the effect of host age on the outcome of TMUV infection. For each age group, clinical signs, gross and microscopic lesions, viral copy numbers in tissues and serum neutralizing antibody titers were recorded. Age-related differences in the resistance to TMUV infection were observed with younger ducks being more susceptible. Some ducks infected at 5 day and 2 week of age developed severe clinical signs, including severe neurological dysfunction and death. However, subclinical signs and no mortality were observed in ducks infected at 5 week of age. A decline in the severity of gross and microscopic lesions was observed as ducks mature. Systemic infections were established in the three age groups post challenge. Higher viral copy numbers in the tissues, especially in vital organs such as the brain and the heart, were developed in the ducks infected at 5 day of age than older ducks, correlating with the severity of clinical signs and lesions in the tissues. Furthermore, ducks infected at 5 week of age developed significantly higher serum neutralizing antibody titers than ducks infected at 5 day of age as determined by serum neutralization test. Therefore, age-related differences in the resistance to TMUV infection should be considered when studying the pathogenicity, pathogenesis, formulation of the vaccination and therapy strategies of TMUV infection in ducks.

Development and Validation of One-Step SYBR Green Real-Time RT-PCR for the Rapid Detection of Newly Emerged Duck Tembusu Virus

Duck Tembusu virus (DTMUV) is a single-stranded positive-sense RNA virus that causes disease to emerge in duck flocks and results in huge economic losses to the duck industry. However, no vaccines and control measures are available in China to date. Development of reliable and fast detection methods is necessary to prevent and control this disease. Therefore, a one-step SYBR Green real-time reverse transcription polymerase chain reaction (RT-PCR) method is established here for DTMUV detection. The results show that the method can specifically detect DTMUV without cross-reactions with selected avian pathogens. The sensitivity of the assay was 1000 times greater than that of a conventional RT-PCR and able to test as few as 20 copies from RNA standard samples. The coefficients of variations of inter- and intra-assay values ranged from 0.09% to 0.36% and 0.1% to 0.23%, respectively. Testing 168 field samples and 96 experimentally infected samples by conventional RT-PCR and the one-step SYBR Green real-time RT-PCR, the positive rates were 35.1% and 73.8% from field samples and 30.2% and 64.6% from infected samples. The one-step SYBR Green real-time RT-PCR developed in this study was shown to be a sensitive, specific, high-throughput, cost-effective, and simple diagnostic tool for the rapid detection and epidemiological surveillance of the emerging DTMUV infection.

Comparison of real-time reverse transcription loop-mediated isothermal amplification and real-time reverse transcription polymerase chain reaction for duck Tembusu virus

Duck Tembusu virus (DTMUV) has caused huge losses to the poultry industry in China since the spring of 2010. The development of a rapid, convenient, and reliable method to diagnose this emerging duck infectious disease is critical. In the present study, a real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was compared with the real-time reverse transcription polymerase chain reaction (RT-PCR) for detection of DTMUV. The sensitivity of real-time RT-LAMP was equal to that of the real-time RT-PCR, with a detection limit of 0.01 ELD(50) (50% egg lethal dose). The specificity of the real-time RT-LAMP and real-time RT-PCR was confirmed using RNAs and DNAs extracted from related viruses which cause duck infections. The reproducibility of the real-time RT-PCR assay was better than that of the real-time RT-LAMP. Only three results from 96 tissue samples differed between the real-time RT-LAMP and this real-time RT-PCR, confirming the reliability of these methods. This study indicated that the real-time RT-LAMP is simpler, less time-consuming, and more convenient than the real-time RT-PCR. With its high sensitivity, specificity, and convenience, the real-time RT-LAMP is a practical molecular diagnostic method for rapid and quantitative detection of DTMUV infection in a resource-limited setting.

Rapid Detection of Tembusu Virus by Reverse-Transcription, Loop-mediated Isothermal Amplification (RT-LAMP)

A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of Tembusu virus (TMUV) infection. The reaction was performed in one step in a single tube at 64°C for 45 min, with SYBR Green I dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10 copies/μl, and no cross-reaction with other avian viruses was observed. The assay was evaluated further for the diagnosis of TMUV in field samples and compared with conventional RT-PCR, demonstrating that results of the RT-LAMP assay corresponded to those of conventional RT-PCR. In conclusion, RT-LAMP with SYBR Green I dye was shown to be a sensitive, simple assay for the rapid diagnosis of TMUV infection in ducks.