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.

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.