scholarly journals In Vivo Inhibition of Marek’s Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4

2021 ◽  
Vol 9 (1) ◽  
pp. 164
Author(s):  
Arjun Challagulla ◽  
Kristie A. Jenkins ◽  
Terri E. O’Neil ◽  
Shunning Shi ◽  
Kirsten R. Morris ◽  
...  
Keyword(s):  
Chicken Embryo Fibroblast ◽  
Marek's Disease ◽  
Recombinant Vaccines ◽  
Marek’S Disease ◽  
Transgenic Chicken ◽  
Guide Rnas ◽  
Specific Virus ◽  
Transgenic Chickens ◽  
Important Disease

Marek’s disease (MD), caused by MD herpesvirus (MDV), is an economically important disease in chickens. The efficacy of the existing vaccines against evolving virulent stains may become limited and necessitates the development of novel antiviral strategies to protect poultry from MDV strains with increased virulence. The CRISPR/Cas9 system has emerged as a powerful genome editing tool providing an opportunity to develop antiviral strategies for the control of MDV infection. Here, we characterized Tol2 transposon constructs encoding Cas9 and guide RNAs (gRNAs) specific to the immediate early infected-cell polypeptide-4 (ICP4) of MDV. We generated transgenic chickens that constitutively express Cas9 and ICP4-gRNAs (gICP4) and challenged them via intraabdominal injection of MDV-1 Woodlands strain passage-19 (p19). Transgenic chickens expressing both gRNA/Cas9 had a significantly reduced replication of MDV in comparison to either transgenic Cas9-only or the wild-type (WT) chickens. We further confirmed that the designed gRNAs exhibited sequence-specific virus interference in transgenic chicken embryo fibroblast (CEF) expressing Cas9/gICP4 when infected with MDV but not with herpesvirus of turkeys (HVT). These results suggest that CRISPR/Cas9 can be used as an antiviral approach to control MDV infection in chickens, allowing HVT to be used as a vector for recombinant vaccines.

scholarly journals Inhibitory Effects of Nitric Oxide and Gamma Interferon on In Vitro and In Vivo Replication of Marek's Disease Virus

2000 ◽  
Vol 74 (8) ◽  
pp. 3605-3612 ◽  
Author(s):  
Zheng Xing ◽  
Karel A. Schat
Keyword(s):  
Nitric Oxide ◽  
Chicken Embryo Fibroblast ◽  
Gamma Interferon ◽  
Marek's Disease ◽  
No Production ◽  
Dependent Manner ◽  
Marek’S Disease ◽  
Dose Dependent

ABSTRACT The replication of Marek's disease herpesvirus (MDV) and herpesvirus of turkeys (HVT) in chicken embryo fibroblast (CEF) cultures was inhibited by the addition ofS-nitroso-N-acetylpenicillamine, a nitric oxide (NO)-generating compound, in a dose-dependent manner. Treatment of CEF culture, prepared from 11-day-old embryos, with recombinant chicken gamma interferon (rChIFN-γ) and lipopolysaccharide (LPS) resulted in production of NO which was suppressed by the addition ofN G-monomethyl l-arginine (NMMA), an inhibitor of inducible NO synthase (iNOS). Incubation of CEF cultures for 72 h prior to treatment with rChIFN-γ plus LPS was required for optimal NO production. Significant differences in NO production were observed in CEF derived from MDV-resistant N2a (major histocompatibility complex [MHC],B 21 B 21) and MDV-susceptible S13 (MHC,B 13 B 13) and P2a (MHC,B 19 B 19) chickens. N2a-derived CEF produced NO earlier and at higher levels than CEF from the other two lines. The lowest production of NO was detected in P2a-derived CEF. NO production in chicken splenocyte cultures followed a similar pattern, with the highest levels of NO produced in cultures from N2a chickens and the lowest levels produced in cultures from P2a chickens. Replication of MDV and HVT was significantly inhibited in CEF cultures treated with rChIFN-γ plus LPS and producing NO. The addition of NMMA to CEF treated with rChIFN-γ plus LPS reduced the inhibition. MDV infection of chickens treated withS-methylisothiourea, an inhibitor of iNOS, resulted in increased virus load compared to nontreated chickens. These results suggest that NO may play an important role in control of MDV replication in vivo.

Electron Microscopy of a Herpesvirus Isolated from Marek's Disease in Duck and Chicken Embryo Fibroblast Cultures

1968 ◽  
Vol 26 ◽  
pp. 222-223 ◽  
Author(s):  
Keyvan Nazerian
Keyword(s):  
Inclusion Bodies ◽  
Chicken Embryo Fibroblast ◽  
Marek's Disease ◽  
Marek’S Disease ◽  
Duck Embryo Fibroblast ◽  
Multinucleated Cells ◽  
Viral Particles ◽  
Infected Cells ◽  
And Control ◽  
Do So

A herpes-like virus has been isolated from duck embryo fibroblast (DEF) cultures inoculated with blood from Marek's disease (MD) infected birds. Cultures which contained this virus produced MD in susceptible chickens while virus negative cultures and control cultures failed to do so. This and other circumstantial evidence including similarities in properties of the virus and the MD agent implicate this virus in the etiology of MD.Histochemical studies demonstrated the presence of DNA-staining intranuclear inclusion bodies in polykarocytes in infected cultures. Distinct nucleo-plasmic aggregates were also seen in sections of similar multinucleated cells examined with the electron microscope. These aggregates are probably the same as the inclusion bodies seen with the light microscope. Naked viral particles were observed in the nucleus of infected cells within or on the edges of the nucleoplasmic aggregates. These particles measured 95-100mμ, in diameter and rarely escaped into the cytoplasm or nuclear vesicles by budding through the nuclear membrane (Fig. 1). The enveloped particles (Fig. 2) formed in this manner measured 150-170mμ in diameter and always had a densely stained nucleoid. The virus in supernatant fluids consisted of naked capsids with 162 hollow, cylindrical capsomeres (Fig. 3). Enveloped particles were not seen in such preparations.

scholarly journals Identification of the Neoplastically Transformed Cells in Marek's Disease Herpesvirus-Induced Lymphomas: Recognition by the Monoclonal Antibody AV37

2002 ◽  
Vol 76 (14) ◽  
pp. 7276-7292 ◽  
Author(s):  
Shane C. Burgess ◽  
T. Fred Davison
Keyword(s):  
Monoclonal Antibody ◽  
T Cell ◽  
Lymphoma Cell ◽  
Marek's Disease ◽  
Transformed Cells ◽  
Marek’S Disease ◽  
Lymphoma Cells ◽  
Transformed Cell Lines ◽  
First Time

ABSTRACT Understanding the interactions between herpesviruses and their host cells and also the interactions between neoplastically transformed cells and the host immune system is fundamental to understanding the mechanisms of herpesvirus oncology. However, this has been difficult as no animal models of herpesvirus-induced oncogenesis in the natural host exist in which neoplastically transformed cells are also definitively identified and may be studied in vivo. Marek's disease (MD) herpesvirus (MDV) of poultry, although a recognized natural oncogenic virus causing T-cell lymphomas, is no exception. In this work, we identify for the first time the neoplastically transformed cells in MD as the CD4+ major histocompatibility complex (MHC) class Ihi, MHC class IIhi, interleukin-2 receptor α-chain-positive, CD28lo/−, phosphoprotein 38-negative (pp38−), glycoprotein B-negative (gB−), αβ T-cell-receptor-positive (TCR+) cells which uniquely overexpress a novel host-encoded extracellular antigen that is also expressed by MDV-transformed cell lines and recognized by the monoclonal antibody (MAb) AV37. Normal uninfected leukocytes and MD lymphoma cells were isolated directly ex vivo and examined by flow cytometry with MAb recognizing AV37, known leukocyte antigens, and MDV antigens pp38 and gB. CD28 mRNA was examined by PCR. Cell cycle distribution and in vitro survival were compared for each lymphoma cell population. We demonstrate for the first time that the antigen recognized by AV37 is expressed at very low levels by small minorities of uninfected leukocytes, whereas particular MD lymphoma cells uniquely express extremely high levels of the AV37 antigen; the AV37hi MD lymphoma cells fulfill the accepted criteria for neoplastic transformation in vivo (protection from cell death despite hyperproliferation, presence in all MD lymphomas, and not supportive of MDV production); the lymphoma environment is essential for AV37+ MD lymphoma cell survival; pp38 is an antigen expressed during MDV-productive infection and is not expressed by neoplastically transformed cells in vivo; AV37+ MD lymphoma cells have the putative immune evasion mechanism of CD28 down-regulation; AV37hi peripheral blood leukocytes appear early after MDV infection in both MD-resistant and -susceptible chickens; and analysis of TCR variable β chain gene family expression suggests that MD lymphomas have polyclonal origins. Identification of the neoplastically transformed cells in MD facilitates a detailed understanding of MD pathogenesis and also improves the utility of MD as a general model for herpesvirus oncology.

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