Kamran Haq

Induction of innate host responses in the lungs of chickens following infection with a very virulent strain of Marek's disease virus.

The natural route of entry of Mareks disease virus (MDV) is via the respiratory system. However, little is known about host-virus interactions in the lungs. The objective of the present study was to examine MDV replication and induction of innate host responses in the lungs of chickens infected through inhalation. Replication of MDV in lungs was detectable as early as 12 hours post-infection (hpi). The expression of Toll-like receptor (TLR)3 and TLR7 genes was enhanced in response to MDV infection in the lungs. This was associated with the up-regulation of interleukin (IL)-1beta and IL-8 genes. In response to MDV infection, the number of macrophages in lungs of infected chickens was significantly higher compared to uninfected control chickens. The expression of inducible nitric oxide synthase (iNOS) gene was also significantly higher in the lungs at 72 hpi following MDV infection. In conclusion, the present study demonstrates induction of innate host responses to MDV infection in the respiratory system. Further studies are needed to characterize other host responses generated in the lungs following MDV infection.

In vivo administration of ligands for chicken toll-like receptors 4 and 21 induces the expression of immune system genes in the spleen

Toll-like receptors (TLRs) are a group of conserved proteins that play an important role in pathogen recognition in addition to the initiation and regulation of innate and adaptive immune responses. To date, several TLRs have been identified in chickens, each recognizing different ligands. TLR stimulation in chickens has been shown to play a role in host-responses to pathogens. However, the mechanisms through which TLRs modulate the chicken immune system have not been well examined. The present study was conducted to characterize the kinetics of responses to TLR4 and TLR21 stimulation in chickens following intramuscular injections of their corresponding ligands, lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (ODNs), respectively. To this end, relative expression of cytokine genes in the spleen was determined at 2, 6, 12 and 24 h after injection of TLR ligands. The results indicated that LPS strongly induced the up-regulation of some immune system genes early on in the response to treatment, including interferon (IFN)-γ, interleukin (IL)-10, and IL-1β. Furthermore, treatment with CpG ODN promoted the up-regulation of major histocompatibility complex (MHC)-II, IFN-γ and IL-10. The response to CpG ODN appeared to be somewhat delayed compared to the response to LPS. Moreover, we found a significant increase in IFN-α gene expression in response to LPS but not CpG ODNs. Future studies may be aimed to further characterize the molecular mechanisms of TLR activation in chickens or to exploit TLR agonists as vaccine adjuvants.

Vaccine-induced host responses against very virulent Marek's disease virus infection in the lungs of chickens

The aim of this study was to investigate the kinetics of virus replication and cellular responses in the lungs following infection with Mareks disease virus (MDV) and/or vaccination with herpesvirus of turkey (HVT) via the respiratory route. Chickens vaccinated with HVT and challenged with MDV had a higher accumulation of MDV and HVT genomes in the lungs compared to the chickens that received HVT or MDV alone. This increase in virus load was associated with augmented expression of interferon (IFN)-gamma and interleukin (IL)-10, and increased T cell infiltration. These findings shed more light on the immunological events that occur in the lungs after vaccination or infection with MDV.

Immune responses against Marek's disease virus

Abstract It is more than a century since Mareks disease (MD) was first reported in chickens and since then there have been concerted efforts to better understand this disease, its causative agent and various approaches for control of this disease. Recently, there have been several outbreaks of the disease in various regions, due to the evolving nature of MD virus (MDV), which necessitates the implementation of improved prophylactic approaches. It is therefore essential to better understand the interactions between chickens and the virus. The chicken immune system is directly involved in controlling the entry and the spread of the virus. It employs two distinct but interrelated mechanisms to tackle viral invasion. Innate defense mechanisms comprise secretion of soluble factors as well as cells such as macrophages and natural killer cells as the first line of defense. These innate responses provide the adaptive arm of the immune system including antibody- and cell-mediated immune responses to be tailored more specifically against MDV. In addition to the immune system, genetic and epigenetic mechanisms contribute to the outcome of MDV infection in chickens. This review discusses our current understanding of immune responses elicited against MDV and genetic factors that contribute to the nature of the response.

A Toll-Like Receptor 3 Ligand Enhances Protective Effects of Vaccination Against Marek's Disease Virus and Hinders Tumor Development in Chickens

Mareks disease (MD) is caused by Mareks disease virus (MDV). Various vaccines including herpesvirus of turkeys (HVT) have been used to control this disease. However, HVT is not able to completely protect against very virulent strains of MDV. The objective of this study was to determine whether a vaccination protocol consisting of HVT and a Toll-like receptor (TLR) ligand could enhance protective efficacy of vaccination against MD. Hence, chickens were immunized with HVT and subsequently treated with synthetic double-stranded RNA polyriboinosinic polyribocytidylic [poly(I:C)], a TLR3 ligand, before or after being infected with a very virulent strain of MDV. Among the groups that were HVT-vaccinated and challenged with MDV, the lowest incidence of tumors was observed in the group that received poly(I:C) before and after MDV infection. Moreover, the groups that received a single poly(I:C) treatment either before or after MDV infection were better protected against MD tumors compared to the group that only received HVT. No association was observed between viral load, as determined by MDV genome copy number, and the reduction in tumor formation. Overall, the results presented here indicate that poly(I:C) treatment, especially when it is administered prior to and after HVT vaccination, enhances the efficacy of HVT vaccine and improves protection against MDV.

Transcriptome and proteome profiling of host responses to Marek's disease virus in chickens.

Mareks disease (MD) is an immunosuppressive and proliferative disease of domestic chickens caused by a highly oncogenic cell-associated alpha-herpesvirus, named Mareks disease virus (MDV). Despite the availability of highly efficacious vaccines for control of MD and existence of lines of chickens which display differential genetic susceptibility or resistance to this disease, little is known about the underlying mechanisms of MDV-host interactions. The recent advent of global or targeted gene and protein expression profiling has paved the way towards gaining a better understanding of host responses to MDV. The main objective of this review is to discuss some of the recent advancements made in relation to elucidating the mechanisms of MDV pathogenesis, host responses to MDV, genetic resistance/susceptibility to MD, and immunity conferred by vaccines. In this regard, particular emphasis has been placed on studies employing proteome and transcriptome profiling approaches. Finally, the utility of microRNA and RNA interference (RNAi) technologies for functional analysis of genes, proteins, and pathways that play a role in the complex interactions between MDV and its host is discussed.

Establishment of an aerosol-based Marek's disease virus infection model.

Abstract Mareks disease virus (MDV), which is the causative agent of Mareks disease (MD), is shed by infected chickens and transmitted to other chickens through the respiratory route. Experimental reproduction of MD has been commonly done either by intra-abdominal inoculation of cell-associated MDV or by exposure to MDV-infected ‘seeder’ chickens. The former method does not mimic the natural route of MDV infection, whereas the latter method suffers from lack of uniformity in the timing and amount of virus transmission from seeder chickens to susceptible birds. The aim of the present study was to establish an infection model of MDV that mimics the natural route of infection. Here we report that when chickens were exposed for 20 min to aerosols (particle size 1.91 µm) of cell-free MDV suspensions containing 1280 plaque-forming units/ml, which were generated using a nebulizer, pathological and clinical signs of MD were observed in 95%–100% of the aerosol-exposed chickens by 21 days post-infection (dpi). Chickens that were exposed to aerosols and sampled at 1, 2, 3, 10, and 21 dpi showed MDV replication as early as 1 dpi in lungs as well as in other tissues such as spleen and bursa of Fabricius. This infection model will facilitate the studies directed to elucidate MDV-host interaction at the site of virus entry.

Immunity to Marek’s disease: Where are we now?

Mareks disease (MD) in chickens was first described over a century ago and the causative agent of this disease, Mareks disease virus (MDV), was first identified in the 1960s. There has been extensive and intensive research over the last few decades to elucidate the underlying mechanisms of the interactions between the virus and its host. We have also made considerable progress in terms of developing efficacious vaccines against MD. The advent of the chicken genetic map and genome sequence as well as development of approaches for chicken transcriptome and proteome analyses, have greatly facilitated the process of illuminating underlying genetic mechanisms of resistance and susceptibility to disease. However, there are still major gaps in our understanding of MDV pathogenesis and mechanisms of host immunity to the virus and to the neoplastic events caused by this virus. Importantly, vaccines that can disrupt virus transmission in the field are lacking. The current review explores mechanisms of host immunity against Mareks disease and makes an attempt to identify the areas that are lacking in this field.

Influence of vaccination with CVI988/Rispens on load and replication of a very virulent Marek's disease virus strain in feathers of chickens.

Several highly efficacious vaccines are currently available for control of Mareks disease, a lymphoproliferative disease in chickens. However, these vaccines are unable to prevent infection with Mareks disease virus (MDV) in vaccinated birds. This leads to shedding of virulent MDV from feather follicle epithelium and skin epithelial cells of vaccinated and infected chickens. The objective of the present study was to study the interactions between a vaccine strain (CVI988/Rispens) and a very virulent strain of MDV (RB1B) in feathers. We examined genome load and replication of CVI988 and MDV-RB1B strains at various time points post infection. Moreover, we evaluated cytokine expression in feathers as indicators of immunity generated in response to vaccines against MDV. Analysis of feathers collected between 4 and 21 days post infection (d.p.i.) revealed a steady level of CVI988 genome load in the presence or absence of RB1B. Infection with MDV resulted in a significant increase in RB1B genome load peaking at 14 d.p.i. Importantly, vaccination with CVI988 resulted in a significant reduction in accumulation of MDV-RB1B in feathers. RB1B genome accumulation in feather tips was associated with increased expression of interferon-α at 14 d.p.i. and interferon-Sγ at earlier time points, 4 and 7 d.p.i. compared with 10 and 14 d.p.i. Interleukin-10 and interleukin-6 were up-regulated at 14 d.p.i. in the infected groups. This study expands our understanding of the dynamics of replication of vaccine and virulent MDV strains in the feathers and illuminates mechanisms associated with immunity to Mareks disease.

Interferon-γ influences immunity elicited by vaccines against very virulent Marek's disease virus.

Vaccination of chickens with herpesvirus of turkey (HVT) confers only partial protection against challenge with a very virulent Mareks disease virus (MDV). Here, we evaluated the ability of recombinant chicken interferon-gamma (rChIFN-γ) to enhance protective efficacy of HVT against the very virulent MDV strain, RB1B. The bioactivity of IFN-γ expressed by a plasmid expression vector was confirmed by its ability to stimulate a chicken macrophage cell line (HD11) to produce nitric oxide (NO) in vitro. The administration of HVT with 5μg of pcDNA:chIFN-γ plasmid reduced the incidence of tumor development significantly when compared to vaccinated birds (77.7% in the HVT+empty vector group and 80% in HVT group versus 33.3% in the HVT+chIFN-γ group) and significantly increased IFN-γ expression in the splenocytes of the protected group, suggesting that rChIFN-γ increases the potency of HVT against MDV. Further analysis demonstrated that the protected birds that received HVT vaccine and/or plasmid had lower MDV genome load and lower amounts of transcripts for meq and vIL-8 than in the birds without lesions. Similarly, lower expression of IL-10, IL-18 and IL-6 was observed in the chickens without lesions compared to the chickens that had lesions, suggesting an inverse association between up-regulation of these cytokines and vaccine-induced immunity. In conclusion, IFN-γ can positively influence immunity conferred by HVT vaccination against challenge with a very virulent Mareks disease virus (vvMDV) in chickens.