Sanjay M. Reddy

Rescue of a pathogenic Marek's disease virus with overlapping cosmid DNAs: Use of a pp38 mutant to validate the technology for the study of gene function

Mareks disease virus (MDV) genetics has lagged behind that of other herpesviruses because of the lack of tools for the introduction of site-specific mutations into the genome of highly cell-associated oncogenic strains. Overlapping cosmid clones have been successfully used for the introduction of mutations in other highly cell-associated herpesviruses. Here we describe the development of overlapping cosmid DNA clones from a very virulent oncogenic strain of MDV. Transfection of these cosmid clones into MDV-susceptible cells resulted in the generation of a recombinant MDV (rMd5) with biological properties similar to the parental strain. To demonstrate the applicability of this technology for elucidation of gene function of MDV, we have generated a mutant virus lacking an MDV unique phosphoprotein, pp38, which has previously been associated with the maintenance of transformation in MDV-induced tumor cell lines. Inoculation of Mareks disease-susceptible birds with the pp38 deletion mutant virus (rMd5Δpp38) revealed that pp38 is involved in early cytolytic infection in lymphocytes but not in the induction of tumors. This powerful technology will speed the characterization of MDV gene function, leading to a better understanding of the molecular mechanisms of MDV pathogenesis. In addition, because Mareks disease is a major oncogenic system, the knowledge obtained from these studies may shed light on the oncogenic mechanisms of other herpesviruses.

Use of avian bornavirus isolates to induce proventricular dilatation disease in conures

The fulfillment of Koch’s postulates shows that the virus causes proventricular dilatation disease in parrots.

Identification of differentially expressed miRNAs in chicken lung and trachea with avian influenza virus infection by a deep sequencing approach

BackgroundMicroRNAs (miRNAs) play critical roles in a wide spectrum of biological processes and have been shown to be important effectors in the intricate host-pathogen interaction networks. Avian influenza virus (AIV) not only causes significant economic losses in poultry production, but also is of great concern to human health. The objective of this study was to identify miRNAs associated with AIV infections in chickens.ResultsTotal RNAs were isolated from lung and trachea of low pathogenic H5N3 infected and non-infected SPF chickens at 4 days post-infection. A total of 278,398 and 340,726 reads were obtained from lung and trachea, respectively. And 377 miRNAs were detected in lungs and 149 in tracheae from a total of 474 distinct chicken miRNAs available at the miRBase, respectively. Seventy-three and thirty-six miRNAs were differentially expressed between infected and non-infected chickens in lungs and tracheae, respectively. There were more miRNAs highly expressed in non-infected tissues than in infected tissues. Interestingly, some of these differentially expressed miRNAs, including miR-146, have been previously reported to be associated with immune-related signal pathways in mammals.ConclusionTo our knowledge, this is the first study on miRNA gene expression in AIV infected chickens using a deep sequencing approach. During AIV infection, many host miRNAs were differentially regulated, supporting the hypothesis that certain miRNAs might be essential in the host-pathogen interactions. Elucidation of the mechanism of these miRNAs on the regulation of host-AIV interaction will lead to the development of new control strategies to prevent or treat AIV infections in poultry.

Characterization of the Chromosomal Binding Sites and Dimerization Partners of the Viral Oncoprotein Meq in Marek's Disease Virus-Transformed T Cells

ABSTRACT Mareks disease virus (MDV) is an acute transforming alphaherpesvirus that causes T-cell lymphomas in chickens. We previously reported the identification of a putative oncogene, meq, that is encoded only by the oncogenic serotype of MDV. The gene product, Meq, is a latent protein that is consistently expressed in MDV-transformed lymphoblastoid cells and tumor cells. Meq has a bZIP (basic leucine zipper) structure resembling the family of Jun/Fos. The mechanism whereby Meq transforms T cells remains poorly understood. In this study, we explored the properties of Meq as a transcriptional factor. We analyzed Meqs dimerization partners and its target genes in MSB-1, an MDV-transformed T-cell line. By using in vitro assays, we first demonstrated Meqs potential to dimerize with a variety of bZIP proteins. We then identified c-Jun as the primary dimerization partner of Meq. Both are found to be colocalized in the nucleus and corecruited to promoters with AP-1 sequences. By using chromatin immunoprecipitation (ChIP), we scanned the entire MDV genome for Meq binding sites and found three regions that were enriched with Meq binding: the MDV lytic replication origin, the promoter for Meq, and the promoter for ICP4. Transactivation assays using the above promoters showed that Meq/Meq homodimers exhibited repression activity, whereas Meq/Jun heterodimers showed activation. Finally, we were able to show by ChIP that Meq is recruited to the interleukin-2 promoter in a region encompassing an AP-1 site. Thus, in addition to providing general knowledge about the transcriptional properties of Meq, our studies revealed for the first time the ability of Meq to interact with the latent MDV and host genomes. Our data suggest, therefore, a role for Meq in viral genome regulation during latency, in addition to its putative causal role in T-cell transformation.

Marek's Disease Virus-Encoded vIL-8 Gene Is Involved in Early Cytolytic Infection but Dispensable for Establishment of Latency

ABSTRACT Mareks disease, a lymphoproliferative disease of chickens, is caused by an alphaherpesvirus, Mareks disease virus (MDV). This virus encodes a virokine, vIL-8, with general homology to cellular CXC chemokines such as interleukin-8 (IL-8) and Gro-α. To study the function of vIL-8 gene, we deleted both copies of vIL-8 residing in the terminal repeat long and internal repeat long region of the viral genome and generated a mutant virus with vIL-8 deleted, rMd5/ΔvIL-8. Growth kinetics study showed that vIL-8 gene is dispensable for virus replication in cell culture. In vivo, the vIL-8 gene is involved in early cytolytic infections in lymphoid organs, as evidenced by limited viral antigen expression of rMd5/ΔvIL-8. However, the rMd5/ΔvIL-8 virus is unimpaired in virus replication in the feather follicle epithelium. vIL-8 does not appear to be important for establishment of latency, since rMd5/ΔvIL-8 and the wild-type virus have similar viremia titers at 14 days postinfection, a period when the virus titer comes primarily from reactivated latent genomes. Nevertheless, because of the impaired cytolytic infections, the overall transformation efficiency of the virus with vIL-8 deleted is much lower, as reflected by the reduced number of transformed cells at 5 weeks postinoculation and the presence of fewer gross tumors. Importantly, the revertant virus that restored the expression of vIL-8 gene also restored the wild-type phenotype, indicating the deficient phenotypes are results of vIL-8 deletion. One of the interesting differences between the MDV vIL-8 gene and its cellular counterpart is the presence of a DKR (Asp-Lys-Arg) motif instead of ELR (Glu-Leu-Arg) preceding the invariable CXC motif. To study the significance of this variation, we generated recombinant MDV, rMd5/vIL-8-ELR, carrying the ELR motif. Both in vitro and in vivo studies revealed that the DKR motif is as competent as ELR in pathogenesis of MDV.

Glycoproteins E and I of Marek's Disease Virus Serotype 1 Are Essential for Virus Growth in Cultured Cells

ABSTRACT The role of glycoprotein E (gE) and gI of Mareks disease virus serotype 1 (MDV-1) for growth in cultured cells was investigated. MDV-1 mutants lacking either gE (20ΔgE), gI (20ΔgI), or both gE and gI (20ΔgEI) were constructed by recE/T-mediated mutagenesis of a recently established infectious bacterial artificial chromosome (BAC) clone of MDV-1 (D. Schumacher, B. K. Tischer, W. Fuchs, and N. Osterrieder, J. Virol. 74:11088–11098, 2000). Deletion of either gE or gI, which form a complex in MDV-1-infected cells, resulted in the production of virus progeny that were unable to spread from cell to cell in either chicken embryo fibroblasts or quail muscle cells. This was reflected by the absence of virus plaques and the detection of only single infected cells after transfection, even after coseeding of transfected cells with uninfected cells. In contrast, growth of rescuant viruses, in which the deleted glycoprotein genes were reinserted by homologous recombination, was indistinguishable from that of parental BAC20 virus. In addition, the 20ΔgE mutant virus was able to spread from cell to cell when cotransfected into chicken embryo fibroblasts with an expression plasmid encoding MDV-1 gE, and the 20ΔgI mutant virus exhibited cell-to-cell spread capability after cotransfection with a gI expression plasmid. The 20ΔgEI mutant virus, however, was not able to spread in the presence of either a gE or gI expression plasmid, and only single infected cells were detected by indirect immunofluorescence. The results reported here demonstrate for the first time that both gE and gI are absolutely essential for cell-to-cell spread of a member of the Alphaherpesvirinae.

Integrated analysis of microRNA expression and mRNA transcriptome in lungs of avian influenza virus infected broilers

BackgroundAvian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited.ResultsTotal RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6 M and 3.3 M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher’s exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44 K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection.ConclusionsA comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122–1, 122–2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken.

Protection and synergism by recombinant fowl pox vaccines expressing multiple genes from Marek's disease virus

Abstract Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Mareks disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB1, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB1, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody–positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB1 (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB1UL32 (46%), rFPV/gB1gEgI (72%), and rFPV/gB1gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB1 and rFPV/gI were comparable with that of HVT. Only gB1 and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB1gEgI (72%) and rFPV/gB1gEgIUL32(70%) against Mareks disease was significantly enhanced compared with a single gB1 or gI gene (40%). This protective synergism between gB1 and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB1gIgEUL32 + HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB1gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement.

Marek's Disease Virus Infection in the Brain: Virus Replication, Cellular Infiltration, and Major Histocompatibility Complex Antigen Expression

Mareks disease virus (MDV) infection in the brain was studied chronologically after inoculating 3-week-old chickens of two genetic lines with two strains of serotype 1 MDV representing two pathotypes (v and vv++). Viral replication in the brain was strongly associated with the development of lesions. Three viral antigens (pp38, gB, and meq) were detected in the brain of infected chickens. Marked differences between v and vv++ pathotypes of MDV were identified for level of virus replication, time course of brain lesions, and expression of major histocompatibility complex (MHC) antigens. Two pathologic phenomena (inflammatory and proliferative) were detected in the brain of chickens inoculated with vv+MDV, but only inflammatory lesions were observed in those inoculated with vMDV. Inflammatory lesions, mainly composed of macrophages, CD4+ T cells, and CD8+ T cells, started at 6-10 days postinoculation (dpi) and were transient. Proliferative lesions, characterized by severe infiltrates of CD4+CD8- T cells (blasts), started at 19–26 dpi and persisted. Expression of MHC antigens in endothelial cells and infiltrating cells within the brain was influenced by MDV infection. Upregulation of MHC class II antigen occurred in all treatment groups, although it was more severe in those inoculated with vv+MDV. MHC class I antigen was downregulated only in those groups inoculated with vv+MDV. These results enhance our understanding of the nature and pattern of MDV infection in the brain and help to explain the neurovirulence associated with highly virulent MDV.

Recombinant Marek's disease virus (MDV) lacking the Meq oncogene confers protection against challenge with a very virulent plus strain of MDV

Mareks disease virus (MDV) encodes a basic leucine-zipper protein, Meq, that shares homology with the Jun/Fos family of transcriptional factors. Conclusive evidence that Meq is an oncogene of MDV came from recent studies of a Meq-null virus, rMd5 Delta Meq. This virus replicated well in vitro, but was non-oncogenic in vivo. Further characterization of this virus in vivo indicated that the meq gene is dispensable for cytolytic infection since it replicated well in the lymphoid organs and feather follicular epithelium. Since rMd5 Delta Meq virus was apathogenic for chickens, we set out to investigate whether this virus could be a good candidate vaccine. Vaccine efficacy experiments conducted in Avian Disease and Oncology Laboratory (ADOL) 15I(5)x 7(1) chickens vaccinated with rMd5 Delta Meq virus or an ADOL preparation of CVI988/Rispens indicated that the Meq-null virus provided protection superior to CVI988/Rispens, the most efficacious vaccine presently available, following challenge with a very virulent (rMd5) and a very virulent plus (648A) MDV strains.