Isolation and Characterization of an Isolate (HN) of Marek''s Disease Virus with Low Pathogenicity

A cytopathic agent was isolated and characterized as an isolate of Marek's disease herpesvirus (MDHV) with low pathogenicity, and referred to as the HN isolate. This isolate of MDHV did not cause clinical Marek's disease (MD) or death in a highly susceptible line of chickens within 5 weeks after exposure. Gross lesions of limited extent were noted in a few of the inoculated birds. Microscopic nerve lesions in the inoculated and contact-infected birds were invariably minimal, closely resembling C-type MD lesions.     

Interactions between Marek's Disease Herpesvirus and Avian Leucosis Virus in Tissue Culture

A GROUP B herpesvirus is important in the aetiology of Marek's disease, a highly contagious lymphoproliferative disease of chickens^1,2. Chicks inoculated with enveloped Marek's disease herpesvirus (MDHV), extracted from feather follicle epithelium of chickens with the disease, developed tumour-like aggregates of lymphoid cells in the viscera and frequently in the peripheral nerves^3,4. Cultures of chicken embryo fibroblast (CEF) cells infected with MDHV develop discrete foci of altered cells⁵. Our data show that MDHV infection of cultures of CEF cells, previously infected with an avian leucosis virus (RAV-2), results in both a reduction in the number of MDHV foci and an increase in the complement fixing avian leucosis antigen (COFAL)⁶ titre.     

Radial Immunodiffusion: a Simple and Rapid Method for Detection of Marek's Disease Antigen(s)

A qualitative radial immunodiffusion technique is described which detects antigen(s) in feathers from live or dead chickens infected with Marek''s disease herpesvirus. Antiserum, which is incorporated into a support medium, reacts with antigen(s) in the feather tip producing a radial precipitin ring. Antigen(s) was detected in 93.3% of experimentally inoculated chickens 21 days postinoculation and in 100% of infected birds subsequently tested through 6 weeks. No antigen was detectable in the feathers of uninoculated control chickens. The technique is simple and rapid to perform. Positive tests could be detected after 1 to 2 hours of incubation. Antigen detection by the radial immunodiffusion test correlated well with other criteria of infection. This technique should have application as a laboratory research tool and as an adjunct for a rapid flock diagnosis of Marek''s disease.     

MicroRNA profiling in the bursae of Marek's disease virus-infected resistant and susceptible chicken lines

Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a cell-associated oncogenic alpha-herpesvirus, Marek's disease virus (MDV). Clinical signs of MD include bursal/thymic atrophy, neurologic disorders, and T cell lymphomas. MiRNAs play key roles in regulation of gene expression by targeting translational suppression or mRNA degradation. MDV encodes miRNAs that are associated with viral pathogenicity and oncogenesis. In this study, we performed miRNA sequencing in the bursal tissues, non-tumorous but viral-induced atrophied lymphoid organ, from control and infected MD-resistant and susceptible chickens at 21 days post infection. In addition to some known miRNAs, a minimum of 300 novel miRNAs were identified in each group that mapped to the chicken genome with no sequence homology to existing miRNAs in chicken miRbase. Comparative analysis identified 54 deferentially expressed miRNAs between the chicken lines that might shed light on underlying mechanism of bursal atrophy and resistance or susceptibility to MD.     

Lyophilization of Cell-Free Marek's Disease Herpesvirus and a Herpesvirus from Turkeys

Cell extracts of the JM and GA strains of Marek''s disease herpesvirus and the FC 126 strain of turkey herpesvirus were lyophilized with various stabilizers. Much higher virus titers were obtained with stabilizer than without stabilizer. Titers increased even further in the case of the Marek''s disease virus strains by the addition of a chelating agent, disodium ethylenediaminetetraacetate.     

Enhancement and interference in chickens inoculated with Marek's disease herpesvirus and oncornaviruses.

Enhancement of mortality rates and symptomatology was observed in isolator-held LSI-SPF chickens concurrently inoculated with MDHV and avian oncornaviruses (RAV-1, RAV-2, RAV-7, RAV-50, or REV). Interference with MD antigen production also was demonstrated in extracts of the feather follicle epithelium from chickens inoculated with both MDHV and RAV-1.     

Genetic mapping of quantitative trait loci affecting susceptibility to Marek's disease virus induced tumors in F2 intercross chickens.

Marek''s disease (MD) is a lymphoproliferative disease caused by the MD virus (MDV), which costs the poultry industry nearly $1 billion annually. To identify quantitative trait loci (QTL) affecting MD susceptibility, the inbred lines 6(3) (MD resistant) and 7(2) (MD susceptible) were mated to create more than 300 F2 chickens. The F2 chickens were challenged with MDV JM strain, moderately virulent) at 1 wk of age and assessed for MD susceptibility. The QTL analysis was divided into three stages. In stage 1, 65 DNA markers selected from the chicken genetic maps were typed on the 40 most MD-susceptible and the 40 most MD-resistant F2 chickens, and 21 markers residing near suggestive QTL were revealed by analysis of variance (ANOVA). In stage 2, the suggestive markers plus available flanking markers were typed on 272 F2 chickens, and three suggestive QTL were identified by ANOVA. In stage 3, using the interval mapping program Map Manager and permutation tests, two significant and two suggestive MD QTL were identified on four chromosomal subregions. Three to five loci collected explained between 11 and 23% of the phenotypic MD variation, or 32-68% of the genetic variance. This study constitutes the first report in the domestic chicken on the mapping of non-major histocompatibility complex QTL affecting MD susceptibility.     

The Leeuwenhoek Lecture, 1997. Marek's disease herpesvirus: oncogenesis and prevention.

There are a number of neoplasias for which a herpesvirus is an essential part of the aetiology. Of these, Marek''s disease is the most common and provides excellent opportunities for the study of a herpesvirus-induced tumour both experimentally and under natural conditions in the field. Marek''s disease is caused by an alpha herpesvirus; it differs from the other oncogenic herpesviruses which are gamma herpesviruses. It is a ubiquitous virus in poultry populations of the world and is highly cell-associated and contagious, yet only a proportion of infected fowl develop tumours. Evidence is presented to suggest that at least one of the reasons for a wide variation in the incidence of the disease is a temporal interplay between virulent viruses and viruses of low or no virulence. The viral genes associated with the oncogenicity of Marek''s disease virus (MDV) are discussed and it is concluded that it is likely that several genes are involved. Finally, a brief history of vaccination to control Marek''s disease is given and mode of action discussed. It is concluded that the mechanism of protection is mainly through an antiviral cell mediated immune response, resulting in a lowered challenge virus burden. Marek''s disease viruses over the past 40 years have been evolving greater oncogenicity, some of which are not adequately controlled by the vaccines that are currently available. It is suggested that for MDV to produce tumours, there is a need for the cytolytic infection phase and that infection must be with an MDV which possesses a functional gC, ICP4 for maintaining latency which allows the expression of at least the 1.8 kb family, pp38, meq, and possibly pp14 genes, for maintaining the tumour state and possibly initiating this state. Intervention in this process reduces the chance of tumour formation and incidence in a population which can occur through natural or man-mediated infection with non-pathogenic MDVs.     

Fluorescently tagged pUL47 of Marek's disease virus reveals differential tissue expression of the tegument protein in vivo

Marek''s disease virus (MDV), a lymphotropic alphaherpesvirus, causes Marek''s disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo, with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo, vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo, while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo, as well as production of free virus in FFE cells.     

Neutralization Studies with Marek's Disease Virus and Turkey Herpesvirus

Use of Marek''s disease virus (MDV) in a neutralization test presents several problems, which are described, making this potentially useful test difficult. To obviate these difficulties, a plaque reduction test has been designed based on cross-neutralization of turkey herpesvirus (HVT) by serum-neutralizing MDV. The technique for such a neutralization test is outlined. Kinetics of development of neutralizing antibodies in chickens inoculated with HVT and MDV are described. The neutralization test can be used to evaluate viability of HVT vaccines and the possible role of neutralizing antibodies in the protection afforded by vaccination against MDV-induced tumors.     

A genome‐wide SNP scan reveals two loci associated with the chicken resistance to Marek's disease

Marek's disease (MD) is a neoplastic disease in chickens, caused by the Marek's disease virus (MDV). To investigate host genetic resistance to MD, we conducted a genome‐wide association study (GWAS) on 67 MDV‐infected chickens based on a case and control design, including 57 susceptible chickens in the case group and 10 resistant chickens as controls. After searching 38 655 valid genomic markers, two SNPs were found to be associated with host resistance to MD. One SNP, rs14527240, reaching chromosome‐wide significance level (P < 0.01) was located in the SPARC‐related modular calcium‐binding 1 (SMOC1) gene on GGA5. The other one, GGaluGA156129, reaching genome‐wide significance (P < 0.05), was located in the protein tyrosine phosphatase, non‐receptor type 3 (PTPN3) gene on GGA2. In addition, expression patterns of these two genes in spleens were detected by qPCR. The expression of SMOC1 was significantly up‐regulated (P < 0.05), whereas the expression of PTNP3 did not show significance when the case group was compared with the control group. Up‐regulation of SMOC1 in susceptible spleens suggests its important roles in MD tumorigenesis. This is the first study to investigate MD‐resistant loci, and it demonstrates the power of GWASs for mapping genes associated with MD resistance.     

A Virulent Bioluminescent and Fluorescent Dual-Reporter Marek's Disease Virus Unveils an Alternative Spreading Pathway in Addition to Cell-to-Cell Contact

Marek''s disease virus (MDV) is a growing threat for the poultry industry. Unfortunately, despite successful vaccination against the disease, MDV remains in circulation within vaccinated flocks, leading to the selection of increasingly virulent pathotypes. Detailed knowledge of the virus biology and the host-virus interaction is required to improve the vaccine efficiency. In the present study, I engineered an original, dual-reporter MDV to track and quantify virus replication in vitro and in vivo.     

Highly pathogenic avian influenza in magpies (Pica pica sericea) in South Korea

Highly pathogenic avian influenza (HPAI) is an extremely infectious, systemic viral disease of birds that produces high mortality and morbidity. HPAI was diagnosed in the three dead magpies (Pica pica sericea) submitted to the National Veterinary Research and Quarantine Service. At necropsy, the prominent lesions were multifocal or coalescing necrosis of the pancreas with enlargement of the livers and spleens. Microscopically, there were severely necrotizing pancreatitis and lymphocytic meningoencephalitis. Influenza viral antigen was also detected in areas closely associated with histologic lesions. Avian influenza virus was isolated from cecal tonsils and feces of the magpies. The isolated virus was identified as a highly pathogenic H5N1, with hemagglutinin proteolytic cleavage site deduced amino acid sequence of QREKRKKR/GLFGAIAG. To determine the pathogenicity of the isolate, eight 6-wk-old specific-pathogen-free chickens were inoculated intravenously with the virus, and all birds died within 24 hr after inoculation. This is the first report of HPAI in magpies.     

Phenotypic and Genotypic Characterization of Avian Escherichia coli O86:K61 Isolates Possessing a Gamma-Like Intimin

Escherichia coli O86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species. Studies in the late 1990s identified E. coli O86:K61 as the cause of mortality in a variety of wild birds, and in this study, 34 E. coli O86:K61 isolates were examined. All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type 1 and curli fimbriae. Thirty-three isolates were positive for the eaeA gene encoding a gamma type of intimin. No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin. Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy. Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model. Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract. No deaths were recorded in inoculated chicks. These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacing E. coli.     

A T-cell antigen system of chickens: Ly-4 and Marek's disease

A search was made for lymphocyte antigens associated with resistance or susceptibility to the T-cell lymphoma induced by the herpes virus of Marek's disease (MD), the experimental model for Burkitt's lymphoma of humans. Antisera were produced by reciprocal immunization with whole blood between an MD-resistant and susceptible line of chickens compatible at the major histocompatibility complex (MHC), and were tested against lymphocytes of both lines. The lymphocytes were not agglutinated, immobilized, or lysed, but their ability to evoke graft-versus-host (GVH) splenomegaly was reduced. This inhibitory activity was line-specific, and these sera had a maximum limiting effect on GVH splenomegaly at a dilution of 1/50 and a minimum at 1/800 dilution. A test based on the differential limitation of GVH splenomegaly by a pair of alloantisera was used to identify the antigens in F₁ and F₂ generations. The segregation results established a locus,Ly-4, with two codominant alleles,Ly- 4^a andLy-4 ^b .Ly-4 is distinct from theA, B, orC blood group loci and from theBu-1 locus determining B-cell antigens, but may be linked to theTh-1 locus determining T-cell antigens (recombination frequency of 32 percent). Tentative evidence was obtained from comparisons of homozygous F₂ and F₃ progeny for association of theLy-4 allele characteristic of the susceptible line with increased incidence of MD.     

Eliminating Tobacco Rattle Virus from Viruliferous Paratrichodorus allius and Establishing a New Virus-Vector Combination

A reliable method to eliminate tobacco rattle virus (TRV) from viruliferous Paratrichodorus allius populations was developed. This virus is vectored by P. allius in the Pacific Northwest and causes corky ringspot disease (CRS) of potato. The viruliferous nematodes that were reared on ''Vernema'' alfalfa or ''770'' scotch spearmint for at least 3 months did not transmit TRV to ''Samsun NN'' tobacco, a suitable indicator plant, and did not cause CRS symptoms on ''Russet Norkotah'' tubers. A new isolate of TRV was introduced into a nonviruliferous population of P. allius. First, tobacco plants were inoculated with a field population of P. allius that transmitted an isolate of TRV that caused severe symptoms on potato. The tobacco roots were then washed free from soil and dipped in 0.525% sodium hypochlorite to remove the initial nematode inoculum. After the disinfected tobacco plants recovered and began to grow, the virus-free population of P. allius was introduced around the root system to acquire the new virus isolate from tobacco roots. The newly established virus-vector combination caused CRS symptoms on ''Russet Norkotah'' that were characteristic of the more virulent virus isolate, indicating that the virus-free P. allius population had reacquired virus.     

Pathogenicity for chicks of line cells from lymphoma of Marek's disease.

Pathogenicity for chicks of the MSB-1 line, a cell line derived from the tumorous tissue of a chick with Marek's disease (MD) and established by Akiyama & Kato, was studied. Five groups, including a control one, of 20 chicks each were inoculated with 1 X 10(3), 1 X 10(4), 1 X 10(5), 1 X 10(6) and no cells of a 180-day culture of the cell line at one day of age. They were housed all together in an isolation unit. An attempt was first made successfully to isolate MD virus (MDV) directly in culture of kidney cells 3 weeks after inoculation. Horizontal infection was first detected 4 weeks after inoculation. From 3 weeks after inoculation on, the disease with almost the same clinical and pathological pictures as the infection with a virulent strain of MDV showed a high incidence. Morbidity was closely related to the number of MSB-1 line cells inoculated. Parenchymal destruction was conspicuous in the central lymphoid organs of four chicks given the largest number of MSB-1 line cells and sacrificed in extremis about 4 weeks after inoculation. Establishment of MD in chicks inoculated with MSB-1 line cells carrying MDV genome seemed to be initiated under the circumstances where the line cells which had come into contact with susceptible cells in the peritoneal cavity released virulent MDV per se. Then host chicks might be infected with MDV and suffer from MD at a high rate. There was no great difference in oncogenic potential between MSB-1 line cells cultivated in vitro for 180 days and virulent MDV serially passaged through one-day-old chicks.     

Efficiency of Commercial Air Filters Against Marek's Disease Virus

In a series of 10 replicate trials with susceptible chickens as indicators of the presence of virus in the air, filter B₁ (Dri Pak, American Air Filter Co.), which has a dust spot rating of 93 to 97%, and filter C (Astrocel), with a dioctyl phathalate aerosol rating of over 99%, completely removed Marek''s disease virus (MDV) from highly contaminated air. However, air passed through filter B₂ (Duracel), with a dust spot rating of 34 to 45%, remained infectious. Thus, an air filter with a dust spot rating of over 93% will effectively remove MDV from the air. The smallest infectious particles in the air are estimated to be greater than 0.3 μm in diameter.     

Characterization of Breast Tumors Using Diffusion Kurtosis Imaging (DKI)

Aim

The aim of this study was to investigate and evaluate the role of magnetic resonance (MR) diffusion kurtosis imaging (DKI) in characterizing breast lesions.

Materials and Methods

One hundred and twenty-four lesions in 103 patients (mean age: 57±14 years) were evaluated by MR DKI performed with 7 b-values of 0, 250, 500, 750, 1,000, 1,500, 2,000 s/mm² and dynamic contrast-enhanced (DCE) MR imaging. Breast lesions were histologically characterized and DKI related parameters—mean diffusivity (MD) and mean kurtosis (MK)—were measured. The MD and MK in normal fibroglandular breast tissue, benign and malignant lesions were compared by One-way analysis of variance (ANOVA) with Tukey''s multiple comparison test. Receiver operating characteristic (ROC) analysis was performed to assess the sensitivity and specificity of MD and MK in the diagnosis of breast lesions.

Results

The benign lesions (n = 42) and malignant lesions (n = 82) had mean diameters of 11.4±3.4 mm and 35.8±20.1 mm, respectively. The MK for malignant lesions (0.88±0.17) was significantly higher than that for benign lesions (0.47±0.14) (P<0.001), and, in contrast, MD for benign lesions (1.97±0.35 (10⁻³ mm²/s)) was higher than that for malignant lesions (1.20±0.31 (10⁻³ mm²/s)) (P<0.001). At a cutoff MD/MK 1.58 (10⁻³ mm²/s)/0.69, sensitivity and specificity of MD/MK for the diagnosis of malignant were 79.3%/84.2% and 92.9%/92.9%, respectively. The area under the curve (AUC) is 0.86/0.92 for MD/MK.

Conclusions

DKI could provide valuable information on the diffusion properties related to tumor microenvironment and increase diagnostic confidence of breast tumors.