Identification of transcripts and protein products of the UL31, UL37, UL46, UL47, UL48, UL49 and US4 gene homologues of avian infectious laryngotracheitis virus.
ABSTRACT In the present study, the transcription and protein expression of seven genes of infectious laryngotracheitis virus (ILTV) were investigated: UL31 and UL37 possess homologues in all known avian and mammalian herpesviruses, whereas UL46-UL49 and US4 are only conserved in most alphaherpesviruses. A peculiarity of the ILTV genome is the translocation of UL47 from the unique long region to a position upstream of US4 within the unique short region. Northern blot analyses revealed that all of the analysed genes were transcribed most abundantly during the late (gamma) phase of replication, but the only true late (gamma2) gene was UL47. Using monospecific rabbit antisera, the protein products of all of the genes could be detected and localized in ILTV-infected cells. Considerable amounts of the UL31, UL47 and UL48 gene products were found in the cell nuclei, whereas the other proteins were restricted largely to the cytoplasm. Like the respective tegument proteins of other herpesviruses, the UL37 and UL46-UL49 gene products of ILTV were incorporated into virus particles, whereas the UL31 protein and the glycoprotein encoded by US4 (gG) were not detectable in purified virions. It was also demonstrated that the UL48 protein of ILTV is able to activate an alphaherpesvirus immediate-early gene promoter, which is also a typical feature of other UL48 homologues. Taken together, these results indicate that the functions of all of the investigated ILTV proteins are related to those of their homologues in other alphaherpesviruses.
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ABSTRACT: Previous studies indicate that the UL31 protein and its homology play similar roles in nuclear egress of all herpesviruses. However, there is no report on the UL31 gene product of DEV. In this study, we expressed and presented the basic properties of the DEV UL31 product. The entire ORF of the UL31 was cloned into pET 32a (+) prokaryotic expression vector. Escherichia coli BL21(DE3) competent cells were transformed with the construct followed by the induction of protein expression by the addition of IPTG. Band corresponding to the predicted sizes (55 kDa) was produced on the SDS-PAGE. Over expressed 6xHis-UL31 fusion protein was purified by nickel affinity chromatography. The DEV UL31 gene product has been identified by using a rabbit polyclonal antiserum raised against the purified protein. A protein of approximate 35 kDa that reacted with the antiserum was detected in immunoblots of DEV-infected cellular lysates, suggesting that the 35 kDa protein was the primary translation product of the UL31 gene. RT-PCR analyses revealed that the UL31 gene was transcribed most abundantly during the late phase of replication. Subsequently, Immunofluorescence analysis revealed that the protein was widespread speckled structures in the nuclei of infected cells. Western blotting of purified virion preparations showed that UL31 was a component of intracellular virions but was absent from mature extracellular virions. Finally, an Immunofluorescence assay was established to study the distribution of the UL31 antigen in tissues of artificially DEV infected ducks. The results showed that the UL31 antigen was primarily located in the cells of digestive organs and immunological organs. In this work, we present the basic properties of the DEV UL31 product. The results indicate that DEV UL31 shares many similarities with its HSV or PRV homolog UL31 and suggest that functional cross-complementation is possible between members of the Alphaherpesvirus subfamily. Furthermore, in vivo experiments with ducks infected with UL31-defective isolates of DEV will also be of importance in order to assess the possible role of the UL31 protein in viral pathogenesis. These properties of the UL31 protein provide a prerequisite for further functional analysis of this gene.Virology Journal 03/2009; 6:19. · 2.34 Impact Factor
Article: Epidemic of infectious laryngotracheitis in Italy: characterization of virus isolates by PCR-restriction fragment length polymorphism and sequence analysis.[show abstract] [hide abstract]
ABSTRACT: Between May 2007 and October 2008, 34 outbreaks of mild to moderate forms of infectious laryngotracheitis (ILT) occurred in commercial broiler flocks in Italy. Affected birds showed watery eyes, conjunctivitis, nasal discharge, reduction of feed and water consumption, and gasping with expectoration of blood-stained mucus. The mortality rate was < 10%. Gross lesions consisted of conjunctivitis, excess of mucus, blood, or presence of diphtheritic membranes in trachea. A real-time PCR assay was performed to confirm the presence of ILT virus (ILTV) DNA in tracheal tissue homogenates. Twenty-three ILTV isolates were propagated on the chorion-allantoic membrane of embryonated chicken eggs showing typical plaques. PCR combined with restriction fragment length polymorphism and gene sequencing of isolates showed a high genetic correlation between field strains and chicken embryo origin vaccines.Avian Diseases 12/2010; 54(4):1172-7. · 1.46 Impact Factor
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ABSTRACT: Using a combination of bioinformation analysis and Dot blot technique, a gene, designated hereafter as the duck enteritis virus (DEV) UL31 gene (GenBank accession number EF643559), was identified from the DEV CHv genomic library. Then, the UL31 gene was cloned and sequenced, which was composed of 933 nucleotides encoding 310 amino acids. Multiple sequence alignment suggested that the UL31 gene was highly conserved in Alphaherpesvirinae and similar to the other herpesviral UL31. Phylogenetic analysis showed that the gene had a close evolutionary relationship with the avian herperviruses, and DEV should be placed into a single cluster within the subfamily Alphaherpesvirinae. Antigen prediction indicated that several potential B-cell epitopes sites located in the UL31 protein. To further study, the UL31 gene was cloned into a pET prokaryotic expression vector and transformed into Escherichia coli BL21 (DE3). A 55 kDa fusion protein was induced by the further culture at 37 degrees C after addition of 0.8 mM IPTG. Polyclonal antibody raised against the recombinant UL31 from rabbit was prepared. A protein about 55 kDa that reacted with the antibody was detected in immunoblots of bacterial proteins, suggesting that the 55 kDa protein was the product of the UL31 gene. Immunofluorescence analysis revealed that the protein was localized in very fine punctate forms in the nuclei of infected cells. Our results may provide some insight for further research about the gene and also enrich the database of herpesvirus.Molecular Biology Reports 06/2009; 37(3):1495-503. · 2.93 Impact Factor