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.

Immunization with a suicidal DNA vaccine expressing the E glycoprotein protects ducklings against duck Tembusu virus.

BACKGROUD: Duck Tembusu virus (DTMUV), a pathogenic flavivirus, emerged in China since 2010 and causing huge economic loss in the Chinese poultry industry. Although several vaccines have been reported to control DTMUV disease, few effective vaccines are available and new outbreaks were continuously reported. Thus, it is urgently to develop a new effective vaccine for prevention of this disease. METHODS: In this study, a suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon and DTMUV E glycoprotein gene was constructed and the efficacy of this new vaccine was assessed according to humoral and cell-mediated immune responses as well as protection against the DTMUV challenge in ducklings. RESULTS: Our results showed that the recombinant SFV replicon highly expressed E glycoprotein in DEF cells. After intramuscular injection of this new DNA vaccine in ducklings, robust humoral and cellular immune responses were observed in all immunized ducklings. Moreover, all ducklings were protected against challenge with the virulent DTMUV AH-F10 strain. CONCLUSIONS: In conclusion, we demonstrate that this suicidal DNA vaccine is a promising candidate facilitating the prevention of DTMUV infection.

Molecular characterization of a novel reovirus isolated from Pekin ducklings in China.

The complete genome sequence of a novel duck orthoreovirus, designated DRV strain TH11(DRV-TH11), was determined and characterized. The DRV-TH11 genome is comprised of 23,417 bp and its genome organization is more similar to that of avian orthoreoviruses (ARVs) of chicken origin than other reoviruses. The results of comparative sequence analysis and dendrograms based on the µB- and σC-encoding genes indicated that TH11 may be derived from the reassortment of ARVs and classic Muscovy duck reovirus (MDRV). A possible recombinant event was identified using the SimPlot program, and it occurred in the M2 segment. The results indicated that reassortment and mutation play a role in the evolution of duck reovirus.

Emerging fatal gout disease in Chinese goslings linked to acute kidney injury induced by novel goose astrovirus infection.

A novel goose astrovirus (GAstV) has broken out across China in recent years, causing widespread damage to the poultry industry. In goslings infected with GAstV, the leading cause of death is visceral gout. However, our understanding of the mechanism of gout formation in GAstV infection is largely inadequate. The aim of this study was to examine the pathogenicity of a GAstV strain and explore the molecular mechanisms of visceral gout caused by viral infection in goslings. The virulent GAstV strain HR2105/1 was effectively isolated from the visceral tissue of goslings in gout-affected areas. The whole genome of the HR2105/1 strain was sequenced and analyzed. Subsequently, we established a gosling gout models with experimental GAstV infection. Finally, we conducted a study on the mechanism of GAstV induced acute kidney injury. Phylogenetic analysis of the complete genome sequence showed that it was closely related to the strain circulating in China since 2016, and it was grouped within the GAstV-1 cluster. The clinical signs were reproduced by experimental infection of healthy goslings with the isolated strain and were found to be similar to those reported in clinical cases. Moreover, the virus exhibits strong renal tropism. Infection with the GAstV strain HR2105/1 was found to cause acute kidney injury, as evidenced by increased levels of uric acid and creatinine as well as severe pathological damage. Mechanistic experiments with Masson and Picrosirius Red staining revealed fibrosis in renal tissues after GAstV infection. Furthermore, TUNEL staining revealed that GAstV infection triggered renal cell apoptosis. Additionally, RT-qPCR revealed that GAstV infection caused an excessive inflammatory response by upregulating the expression of IL-1ß, IL-6, IL-10, TGF-ß, and iNOS in renal tissues. Overall, our findings demonstrate that GAstV infection causes renal damage by inducing renal cell apoptosis, fibrosis, and excessive inflammatory response, which subsequently leads to hyperuricemia and lethal visceral gout formation. This is the first systematic study on the etiology of lethal gout in goslings caused by GAstV infection, and we believe that the findings can guide vaccine development and therapeutic targets for GAstV-associated renal diseases.

Development of a potential diagnostic monoclonal antibody against capsid spike protein VP27 of the novel goose astrovirus.

Goose astrovirus (GAstVs) is an emerging pathogen of goslings that causes fatal gout, kidney hemorrhages, renomegaly, and high mortality. The GAstVs VP27 protein is an important capsid protein and a candidate for the development of diagnostic reagents. The aim of this study was to clone and express the VP27 gene for preparation of a specific monoclonal antibody (mAb). The VP27 protein was expressed and purified in the supernatant of Escherichia coli BL21. Then, the mAb was obtained with the hybridoma technique and named 2AF11. It was differentiated as IgG1 with the help of immunoglobulin subclass tests. This mAb can specifically recognize the VP27 protein in GAstVs-infected cells, as evidenced by western blot analysis and immunofluorescent assay. Furthermore, this mAb could also detect the VP27 protein in GAstVs-infected tissues, as demonstrated by immunohistochemistry. These findings indicate that this mAb has high diagnostic potential. Therefore, the newly produced anti-VP27 mAb, 2AF11, could be a useful tool as a specific diagnostic marker for GAstVs.

Induction of a robust immunity response against novel duck reovirus in ducklings using a subunit vaccine of sigma C protein.

Novel duck reovirus (NDRV) disease emerged in China in 2011 and continues to cause high morbidity and about 5.0 to 50% mortality in ducklings. Currently there are no approved vaccines for the virus. This study aimed to assess the efficacy of a new vaccine created from the baculovirus and sigma C gene against NDRV. In this study, a recombinant baculovirus containing the sigma C gene was constructed, and the purified protein was used as a vaccine candidate in ducklings. The efficacy of sigma C vaccine was estimated according to humoral immune responses, cellular immune response and protection against NDRV challenge. The results showed that sigma C was highly expressed in Sf9 cells. Robust humoral and cellular immune responses were induced in all ducklings immunized with the recombinant sigma C protein. Moreover, 100% protection against lethal challenge with NDRV TH11 strain was observed. Summary, the recombinant sigma C protein could be utilized as a good candidate against NDRV infection.

Protective immune responses in ducklings induced by a suicidal DNA vaccine of the sigma C gene of novel duck reovirus.

A suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon was evaluated for the development of a vaccine against novel duck reovirus (NDRV). The sigma C gene of NDRV was cloned and inserted into pSCA1, an SFV DNA-based replicon vector. After transfected into BHK-21 cells, the antigenicity of the sigma C protein was confirmed using an indirect immunofluorescence and Western blot assay. Immunogenicity was studied in ducklings. The results showed that NDRV-specific antibodies, neutralizing antibodies, IFN-γ and IL-4 were well induced in ducklings. Furthermore, all of the ducklings were protected against challenge with wild-type NDRV.