2A2 protein of DHAV-1 induces duck embryo fibroblasts gasdermin E-mediated pyroptosis.

The duck hepatitis A virus type 1 (DHAV-1) causes rapid death in ducklings by triggering a severe cytokine storm. Pyroptosis is an inflammatory form of programmed cell death that is directly related to an increase in pro-inflammatory cytokine levels. Only a few studies have explored the mechanisms underlying pyroptosis in virus-infected avian cells. In this study, we established an avian infection model in vitro by infecting duck embryo fibroblasts (DEFs) with the virulent DHAV-1 LY0801 strain. DHAV-1 infection induced pyroptosis in the DEFs by activating gasdermin E (GSDME) protein via caspase-3-mediated cleavage. The genes encoding the different structural and non-structural DHAV-1 proteins were cloned into eukaryotic expression plasmids, and the 2A2 protein was identified as the key protein involved in pyroptosis. The HPLC-tandem mass spectrometry (HPLC-MS/MS) and co-immunoprecipitation (Co-IP) analysis established that DHAV-1 2A2 directly interacted with the mitochondrial anti-viral signaling protein (MAVS) both intracellularly and in vitro. Furthermore, we got the results that N-terminal 1-130 aa of 2A2 was involved in the interaction with MAVS and the C-terminal TM domain of MAVS is necessary for the interaction with 2A2 by Co-IP analysis. To our knowledge, this is the first study to reveal that DHAV-1 protein interacts with host proteins to induce pyroptosis. Our findings provide new insights into the molecular pathogenesis of DHAV-1 infection, and a scientific basis for the prevention and treatment of duck viral hepatitis.

The role of duck LGP2 in innate immune response of host anti-tembusu virus.

Laboratory of Genetics and Physiology 2 (LGP2), along with Retinoic Acid Induced Gene-I (RIG-I) and Melanoma Differentiation Associated Gene 5, are members of the retinoic acid-inducible gene-I-like receptors (RLRs) in pattern recognition receptors, playing an important role in the host's innate immunity. Due to lacking a caspase activation and recruitment domain, LGP2 is controversially regarded as a positive or negative regulator in the antiviral response. This study aimed to explore how duck LGP2 (duLGP2) participates in duck innate immunity and its role in countering the duck Tembusu virus (DTMUV). In duck embryo fibroblast cells, the overexpression of duLGP2 significantly reduced the cell's antiviral capacity by inhibiting type I interferon (IFN) production and the expression of downstream IFN-stimulated genes. Conversely, duLGP2 knockdown had the opposite effect. For the first time, we introduced the LGP2 gene fragment into duck embryos using a lentiviral vector to ensure persistent expression and generated gene-edited ducks with LGP2 overexpression. We demonstrated that duLGP2 facilitates DTMUV replication in both in vitro and in vivo experiments, leading to robust inflammatory and antiviral responses. Interestingly, the repressive effects of duLGP2 on type I IFN production were only observed in the early stage of DTMUV infection, with type I IFN responses becoming enhanced as the viral load increased. These results indicate that duLGP2 acts as a negative regulator during the resting state and early stages of DTMUV infection. This study provides a theoretical basis for further research on duck RLRs and developing new anti-DTMUV drugs or vaccine adjuvants.

Oral vaccine of recombinant Lactococcus lactis expressing the VP1 protein of duck hepatitis A virus type 3 induces mucosal and systemic immune responses.

Duck hepatitis A virus (DHAV) is the major pathogen of duck viral hepatitis, which has caused great economic losses to duck breeding industry. As an effective delivery tool for protein antigens, Lactococcus lactis (L. lactis) has been successfully used to stimulate mucosal and systemic immune response. In this study, a recombinant L. lactis named NZ3900-VP1 was constructed, which could express VP1 protein of DHAV type 3 (DHAV-3) by using a nisin-controlled expression (NICE) system. The animal experiment in both mice and ducklings were performed to detect the immune response and protection effect of oral vaccination by the recombinant L. lactis. The results showed that oral vaccination with L. lactis NZ3900-VP1 significantly induced specific anti-VP1 IgG antibodies and mucosal secretory immunoglobulin A (sIgA) of DHAV-3 in mice and ducklings, and cytokines including interleukin-2 (IL-2), interferon gamma (IFN-γ), interleukin-10 (IL-10) and interleukin-4 (IL-4). Notably, the ducklings vaccinated with L. lactis NZ3900-VP1 were effectively protected when facing natural infestation of DHAV-3, which indicated that the recombinant L. lactis could serve as an effective vaccine to prevent DHAV-3 infection in ducklings.

Identification and characterization of a novel nanobody against duck hepatitis A virus type 1.

Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is an acute and highly contagious disease affecting young ducklings. The VP1 protein is one of the major structural proteins of DHAV-1 carries critical epitopes responsible for the induction of neutralizing antibodies. In this study, we have successfully constructed an immune phage display VHHs library against DHAV-1 with the size of 6 × 106 colonies. A nanobody (Nb) against VP1 protein of DHAV-1, named Nb25, was identified from the immunized phage display library. Nb25 could react with the conserved linear B-cell epitope of 174PAPTST179 in DHAV-1 VP1, even though Nb25 showed no neutralizing activity to DHAV-1. To the best of our knowledge, this is the first report about preparation of anti-DHAV-1 Nbs and identification of the specific conserved linear B-cell epitope of DHAV-1 with Nb, which will facilitate the serologic diagnosis of DHAV-1 infection.

The Functional Role of the 3' Untranslated Region and Poly(A) Tail of Duck Hepatitis A Virus Type 1 in Viral Replication and Regulation of IRES-Mediated Translation.

The duck hepatitis A virus type 1 (DHAV-1) is a member of Picornaviridae family, the genome of the virus contains a 5' untranslated region (5' UTR), a large open reading frame that encodes a polyprotein precursor and a 3' UTR followed by a poly(A) tail. The translation initiation of virus proteins depends on the internal ribosome-entry site (IRES) element within the 5' UTR. So far, little information is known about the role of the 3' UTR and poly(A) tail during the virus proliferation. In this study, the function of the 3' UTR and poly(A) tail of DHAV-1 in viral replication and IRES-mediated translation was investigated. The results showed that both 3' UTR and poly(A) tail are important for maintaining viral genome RNA stability and viral genome replication. During DHAV-1 proliferation, at least 20 adenines were required for the optimal genome replication and the virus replication could be severely impaired when the poly (A) tail was curtailed to 10 adenines. In addition to facilitating viral genome replication, the presence of 3' UTR and poly(A) tail significantly enhance IRES-mediated translation efficiency. Furthermore, 3' UTR or poly(A) tail could function as an individual element to enhance the DHAV-1 IRES-mediated translation, during which process, the 3' UTR exerts a greater initiation efficiency than the poly(A)25 tail.

Identification of a conserved neutralizing linear B-cell epitope in the VP1 proteins of duck hepatitis A virus type 1 and 3.

Duck virus hepatitis (DVH), mainly caused by duck hepatitis A virus (DHAV), is a severe disease threaten to duck industry and has worldwide distribution. As the major structural protein, the VP1 protein of DHAV is able to induce neutralizing antibody in ducks. In this study, a monoclonal antibody (mAb) 4F8 against the intact DHAV-1 particles was used to identify the possible epitope in the three serotypes of DHAV. The mAb 4F8 had weak neutralizing activities to both DHAV-1 and DHAV-3, and reacted with the conserved linear B-cell epitopes of (75)GEIILT(80) in DHAV-1 VP1 and (75)GEVILT(80) in DHAV-3 VP1 protein, respectively, while not with DHAV-2 VP1. This was the first report about identification of the common conserved neutralizing linear B-cell epitope of DHAV-1 and DHAV-3, which will facilitate understanding of the antigenic structure of VP1 and the serologic diagnosis of DHAV infection.

Complete genome sequence of a duck hepatitis a virus type 3 identified in eastern China.

We report here the complete genome sequence of a novel duck hepatitis A virus type 3 (DHAV-3) isolated from a dead Cherry Valley duckling in eastern China. The whole genomic nucleotide sequence and polyprotein amino acid sequence of the virus had higher homology with those of Chinese DHAV-3 isolates, medium homology with those of Korean DHAV-3 isolates, and the lowest homology with those of Vietnamese isolate DN2. The result indicated that the genetic evolution of DHAV-3 isolates had obvious geographical features.

Genetic variation of the VP1 gene of the virulent duck hepatitis A virus type 1 (DHAV-1) isolates in Shandong province of China.

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1 (DHAV-1) isolates, the virulence, cross neutralization assays and the complete sequence of the virion protein 1 (VP1) gene of nine virulent DHAV-1 strains, which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008, were tested. The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses (ELD(50)s) and the median lethal doses (LD(50)s), respectively. The results showed that the ELD(50)s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10(6)/mL to 1.44 × 10(7)/mL, while the LD(50)s were 2.39 × 10(5)/mL to 6.15 × 10(6)/mL. Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates, respectively. Compared with other virulent, moderate virulent, attenuated vaccine and mild strains, the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains. There were three hypervariable regions at the C-terminus (aa 158-160, 180-193 and 205-219) and other variable points in VP1 protein, but which didn't cause virulence of DHAV-1 change.

Development of an indirect ELISA for the detection of duck circovirus infection in duck flocks.

Infection with duck circovirus (DuCV) is associated with growth retardation and developmental problems in farmed ducks. To detect DuCV-specific antibody in duck serum, an indirect enzyme-linked immunosorbent assay (iELISA) method was developed using the recombinant capsid protein antigen prepared by cloning the cap (Cap) gene of DuCV FJ0601 strain into pET-32a (+) vector and expressed in Escherichia coli. Using the optimized iELISA method, DuCV-specific antibodies were detected in 157 (12.96%) of 1211 samples obtained from 17 (89.47%) of 19 meat duck flocks aged from 25 to 40 days and in 89 (22.08%) of 403 samples obtained from 9 (75%) of 12 breeder flocks aged from 14 to 61 weeks. These results indicated that the iELISA method is useful for serological diagnosis of DuCV infection and epidemiological investigation.

[Sequence analysis for the complete provial genome of endogenous avian leukosis virus strain SD0501].

The genomic DNA extracted from chicken embryo fibroblasts (CEF) of SPF chickens from three chicken farms was used as template to amplify the ALV proviral DNA by PCR with four pairs of primers, high positive detection rates of gag - gene (29/46), pol - gene (27/46), env - gene (24/46) and LTR fragment (31/46) were achieved. Eight continuous and overlapping fragments were amplified from one DNA sample with 8 pairs of primers according to published sequences, then cloned into the TA vector and se quenced. The complete sequence of the whole genome of ALV strain SD0501 was established and analyzed with DNAstar software. Comparisons of SD0501 sequence with that of other representative endogenous avian virus strains demonstrated that the genomes of ALV were relatively conservative, the nucleotide identity of all the strains was over 99.1%, and env - gene was over 98.5%. However, a low identity was demonstrated among the representative strains of different subgroups, especially, the env - gene showed obvious difference, the corresponding identity was as low as 56.3% - 91.5%.

[Immunogenicity of envelope glycoprotein gene of reticuloendotheliosis virus expressed in insect cell].

A recombinant baculovirus expressing reticuloendotheliosis virus env gene was constructed with Bac-to-Bac Baculovirus Expression Systems. After transfecting the recombinant virus into Sf9 cells for 3 days, REV env can be detected by indirect immunofluorescence antibody assay (IFA) and Western blot with specific monoclonal antibodies of REV. The oil-water emulsion vaccine was then produced using this infected Sf9 cells lycates and inoculated SPF chickens to validate the immunogenicity for REV. The results show that special anti-REV antibody can maintain more than 45 days and resist the infection of REV viruses. This is the first success to induce anti-REV antibody in chickens by none-live viruses.

[Construction of infectious clone of subgroup J avian leukosis virus strain NX0101 and its pathogenicity].

By using PCR, 3 fragments of provirus cDNA of avian leukosis virus (ALV-J) strain NXO101 were amplified from the genomic DNA of ALV-J infected cells,and then combined in the right direction and sequences into recombinant plasmid pALV-J-NX, containing the whole genome of NX0101. After transfection of chicken embryo fibroblast (CEF) cells with plasmid pALV-J-NX DNA, the rescued virus was identified in CEF by indirect fluorescence antibody test with ALV-J specific monoclonal antibody JE9. The rescued virus could replicate in CEF at a titer of 10(5.6)/mL. The chicken experiment demonstrated that the rescued virus was still able to induce tumors in commercial meat-type broilers.