The immune cell landscape and response of Marek's disease resistant and susceptible chickens infected with Marek's disease virus.

Genetically resistant or susceptible chickens to Marek's disease (MD) have been widely used models to identify the molecular determinants of these phenotypes. However, these prior studies lacked the basic identification and understanding of immune cell types that could be translated toward improved MD control. To gain insights into specific immune cell types and their responses to Marek's disease virus (MDV) infection, we used single-cell RNA sequencing (scRNAseq) on splenic cells from MD resistant and susceptible birds. In total, 14,378 cells formed clusters that identified various immune cell types. Lymphocytes, specifically T cell subtypes, were the most abundant with significant proportional changes in some subtypes upon infection. The largest number of differentially expressed genes (DEG) response was seen in granulocytes, while macrophage DEGs differed in directionality by subtype and line. Among the most DEG in almost all immune cell types were granzyme and granulysin, both associated with cell-perforating processes. Protein interactive network analyses revealed multiple overlapping canonical pathways within both lymphoid and myeloid cell lineages. This initial estimation of the chicken immune cell type landscape and its accompanying response will greatly aid efforts in identifying specific cell types and improving our knowledge of host response to viral infection.

Endogenous Avian Leukosis Virus in Combination with Serotype 2 Marek's Disease Virus Significantly Boosted the Incidence of Lymphoid Leukosis-Like Bursal Lymphomas in Susceptible Chickens.

In 2010, sporadic cases of avian leukosis virus (ALV)-like bursal lymphoma, also known as spontaneous lymphoid leukosis (LL)-like tumors, were identified in two commercial broiler breeder flocks in the absence of exogenous ALV infection. Two individual ALV subgroup E (ALV-E) field strains, designated AF227 and AF229, were isolated from two different breeder farms. The role of these ALV-E field isolates in development of and the potential joint impact in conjunction with a Marek's disease virus (MDV) vaccine (SB-1) were further characterized in chickens of an experimental line and commercial broiler breeders. The experimental line 0.TVB*S1, commonly known as the rapid feathering-susceptible (RFS) line, of chickens lacks all endogenous ALV and is fully susceptible to all subgroups of ALV, including ALV-E. Spontaneous LL-like tumors occurred following infection with AF227, AF229, and a reference ALV-E strain, RAV60, in RFS chickens. Vaccination with serotype 2 MDV, SB-1, in addition to AF227 or AF229 inoculation, significantly enhanced the spontaneous LL-like tumor incidence in the RFS chickens. The spontaneous LL-like tumor incidence jumped from 14% by AF227 alone to 42 to 43% by AF227 in combination with SB-1 in the RFS chickens under controlled conditions. RNA-sequencing analysis of the LL-like lymphomas and nonmalignant bursa tissues of the RFS line of birds identified hundreds of differentially expressed genes that are reportedly involved in key biological processes and pathways, including signaling and signal transduction pathways. The data from this study suggested that both ALV-E and MDV-2 play an important role in enhancement of the spontaneous LL-like tumors in susceptible chickens. The underlying mechanism may be complex and involved in many chicken genes and pathways, including signal transduction pathways and immune system processes, in addition to reported viral genes.IMPORTANCE Lymphoid leukosis (LL)-like lymphoma is a low-incidence yet costly and poorly understood disease of domestic chickens. The observed unique characteristics of LL-like lymphomas are that the incidence of the disease is chicken line dependent; pathologically, it appeared to mimic avian leukosis but is free of exogenous ALV infection; inoculation of the nonpathogenic ALV-E or MDV-2 (SB-1) boosts the incidence of the disease; and inoculation of both the nonpathogenic ALV-E and SB-1 escalates it to much higher levels. This study was designed to test the impact of two new ALV-E isolates, recently derived from commercial broiler breeder flocks, in combination with the nonpathogenic SB-1 on LL-like lymphoma incidences in both an experimental egg layer line of chickens and a commercial broiler breeder line of chickens under a controlled condition. Data from this study provided an additional piece of experimental evidence on the potency of nonpathogenic ALV-E, MDV-2, and ALV-E plus MDV-2 in boosting the incidence of LL-like lymphomas in susceptible chickens. This study also generated the first piece of genomic evidence that suggests host transcriptomic variation plays an important role in modulating LL-like lymphoma formation.

Correction to: Two class I genes of the chicken MHC have different functions: BF1 is recognized by NK cells while BF2 is recognized by CTLs.

The Figure 3 in the original version of this article was incorrectly published. In this article the top panel of Figure 3 that describes the amino acid sequence alignment is now added. The original article has been corrected.

Two class I genes of the chicken MHC have different functions: BF1 is recognized by NK cells while BF2 is recognized by CTLs.

The function of the chicken's major histocompatibility complex (MHC or B complex) class I major (BF2) and minor (BF1) glycoproteins is compared for their expression, ability to present viral antigens to cytotoxic T lymphocytes (CTLs), and interaction with natural killer (NK) cells. MHC-restricted CTLs recognized virus antigen in the context of the BF2*21 major glycoprotein but not the BF1*21 minor glycoprotein. Marek's disease virus (MDV), a large DNA virus known to reduce the cell surface expression of class I glycoprotein, reduced the expression of BF2 glycoprotein while BF1glycoprotein expressions are remained as no change or slight increase. In addition, the expression of BF1*21 class I glycoprotein protected target cells from NK cell lysis while the expression of the BF2*21 class I glycoprotein enhanced NK cell lysis of target cells. Therefore, BF1 and BF2 provide two different cellular immune functions; BF1 negatively regulates the NK cell killing activity and BF2 restricts the antigen specific CTL immune response.

Differences in CD8αα and cecal microbiome community during proliferation and late cytolytic phases of Marek's disease virus infection are associated with genetic resistance to Marek's disease.

Marek's disease (MD) is an important neoplastic disease of chickens caused by Marek ': s disease virus (MDV), a highly oncogenic alphaherpesvirus. In this study using two chicken lines, one resistant and another susceptible to MD, splenic T cells and cecal microbiome were profiled to gain a better understanding of primary differences in these lines. The percent of splenic CD4+ T cells were similar regardless of MDV challenge status in both bird lines. In contrast, CD8αα profiles were different (P < 0.005) between chicken lines under naïve status and under MDV challenge, suggesting that CD8αα T cells play a key role in mediating MDV infection. Microbiome composition was different between naïve resistant (Blautia spp.) and susceptible birds (Streptococcus spp.) (P < 0.05) during initial colonization. With MDV challenge, both chicken lines showed lower numbers of beneficial Faecalibacterium spp. and increased number of Lactobacillus spp. Metabolic profiles between naïve chicken types were similar but with MDV challenge, there were differences in metabolism in both chicken lines, with amino acid metabolism impacted in resistant birds and lipid metabolism in susceptible birds. These results provide insights into immune response and potential interplay with the microbiome during infection with an oncogenic virus.

Evaluation of the Protection Efficacy of a Serotype 1 Marek's Disease Virus-Vectored Bivalent Vaccine Against Infectious Laryngotracheitis and Marek's Disease.

Laryngotracheitis (LT) is a highly contagious respiratory disease of chickens that produces significant economic losses to the poultry industry. Traditionally, LT has been controlled by administration of modified live vaccines. In recent years, the use of recombinant DNA-derived vaccines using turkey herpesvirus (HVT) and fowlpox virus has expanded, as they protect not only against the vector used but also against LT. However, HVT-based vaccines confer limited protection against challenge, with emergent very virulent plus Marek's disease virus (vv+MDV). Serotype 1 vaccines have been proven to be the most efficient against vv+MDV. In particular, deletion of oncogene MEQ from the oncogenic vvMDV strain Md5 (BACδMEQ) resulted in a very efficient vaccine against vv+MDV. In this work, we have developed two recombinant vaccines against MD and LT by using BACδMEQ as a vector that carries either the LT virus (LTV) gene glycoprotein B (gB; BACΔMEQ-gB) or LTV gene glycoprotein J (gJ; BACδMEQ-gJ). We have evaluated the protection that these recombinant vaccines confer against MD and LT challenge when administered alone or in combination. Our results demonstrated that both bivalent vaccines (BACΔMEQ-gB and BACδMEQ-gJ) replicated in chickens and were safe to use in commercial meat-type chickens bearing maternal antibodies against MDV. BACΔMEQ-gB protected as well as a commercial recombinant (r)HVT-LT vaccine against challenge with LTV. However, BACδMEQ-gJ did not protect adequately against LT challenge or increase protection conferred by BACΔMEQ-gB when administered in combination. On the other hand, both BACΔMEQ-gB and BACδMEQ-gJ, administered alone or in combination, protected better against an early challenge with vv+MDV strain 648A than commercial strains of rHVT-LT or CVI988. Our results open a new avenue in the development of recombinant vaccines by using serotype 1 MDV as vectors.

The Influence of Major Histocompatibility Complex and Vaccination with Turkey Herpesvirus on Marek's Disease Virus Evolution.

Over the last five decades, the pathogenicity of the Marek's disease virus (MDV) has evolved from the relatively mild strains (mMDV) observed in the 1960s to the more severe very-virulent-plus strains currently observed in today's outbreaks. The use of vaccines to control Marek's disease (MD), but not the infection cycle, is thought to be the major influence on the evolution of MDV. Selection for genetic resistance to MD has also been employed by the industry to control MD in the commercial setting but the role of host genetics on the evolution of MDV has been difficult to investigate in the field. To investigate the influence of vaccination and host resistance we developed a laboratory model to control and assess the effects of virus and animal genetics on MDV evolution. A bacterial artificial chromosome-derived MDV (Md5B40BAC) was used for in vivo passage (IVP) through turkey herpesvirus (HVT)-vaccinated resistant (MHC-B21) and susceptible (MHC-B13) genetic chicken lines. During IVP in the vaccinated susceptible line, the disease incidence increased from 23% MD in the first IVP to 53% MD during the fifth IVP. In the vaccinated resistant line, disease incidence increased from 0% MD during the first IVP to 29% MD during the fifth IVP. Although the IVP isolates remained relatively mild in the vaccinated resistant chicken line (29% MD) they increased from 0% to 63% MD when used to challenge the vaccinated susceptible chickens. There was no corresponding increase in disease incidence when the virus passed in the vaccinated susceptible genetic line was used to challenge the vaccinated resistant line. From this series of experiments we show that a cloned MDV (Md5B40BAC) can be selected by serial IVP to induce greater disease incidence in vaccinated chickens. This increase in disease incidence occurs in both susceptible and resistant chicken lines but is more easily observed in the susceptible line. Not surprisingly, both host genetics and vaccination play a role in selecting for increased MDV virulence. Our results suggest that the progressive increase in MDV virulence is partially masked as it circulates through vaccinated resistant genetic lines, but by applying this virus to less-resistant genetic lines, virus evolution can be clearly observed. We would predict that the introduction of more-resistant genetic lines into a commercial house contaminated with MDV circulating through susceptible lines would be less likely to produce vaccine breaks than placing susceptible lines into a house in which previously the MDV was circulating through resistant genetic lines.

An MHC class I immune evasion gene of Marek׳s disease virus.

Marek׳s disease virus (MDV) is a widespread α-herpesvirus of chickens that causes T cell tumors. Acute, but not latent, MDV infection has previously been shown to lead to downregulation of cell-surface MHC class I (Virology 282:198-205 (2001)), but the gene(s) involved have not been identified. Here we demonstrate that an MDV gene, MDV012, is capable of reducing surface expression of MHC class I on chicken cells. Co-expression of an MHC class I-binding peptide targeted to the endoplasmic reticulum (bypassing the requirement for the TAP peptide transporter) partially rescued MHC class I expression in the presence of MDV012, suggesting that MDV012 is a TAP-blocking MHC class I immune evasion protein. This is the first unique non-mammalian MHC class I immune evasion gene identified, and suggests that α-herpesviruses have conserved this function for at least 100 million years.

Marek's disease virus influences the core gut microbiome of the chicken during the early and late phases of viral replication.

Marek's disease (MD) is an important neoplastic disease of chickens caused by the Marek's disease virus (MDV), an oncogenic alphaherpesvirus. In this study, dysbiosis induced by MDV on the core gut flora of chicken was assessed using next generation sequence (NGS) analysis. Total fecal and cecum-derived samples from individual birds were used to estimate the influence of MDV infection on the gut microbiome of chicken. Our analysis shows that MDV infection alters the core gut flora in the total fecal samples relatively early after infection (2-7 days) and in the late phase of viral infection (28-35 days) in cecal samples, corresponding well with the life cycle of MDV. Principle component analyses of total fecal and cecal samples showed clustering at the early and late time points, respectively. The genus Lactobacillus was exclusively present in the infected samples in both total fecal and cecal bird samples. The community colonization of core gut flora was altered by viral infection, which manifested in the enrichment of several genera during the early and late phases of MDV replication. The results suggest a relationship between viral infection and microbial composition of the intestinal tract that may influence inflammation and immunosuppression of T and B cells in the host.

The influence of host genetics on Marek's disease virus evolution.

Since the first report of a polyneuritis in chickens by Joseph Marek in 1907, the clinical nature of the disease has changed. Over the last five decades, the pathogenicity of the Marek's disease virus (MDV) has continued to evolve from the relatively mild strains observed in the 1960s to the more severe strains labeled very virulent plus currently observed in today's outbreaks. To understand the influence of host genetics, specifically the major histocompatibility complex (MHC), on virus evolution, a bacterial artificial chromosome-derived MDV (Md5B40BAC) was passed in vivo through resistant (MHC-B21) and susceptible (MHC-B13) Line 0 chickens. Criteria for selecting virus isolates for in vivo passage were based on virus replication in white blood cells 21 days after challenge and evaluation of MD pathology at necropsy. In the MHC-B13-susceptible line the Md5B40BAC virulence consistently increased from 18% Marek's disease (MD) after in vivo passage 1 (B13-IVP1 Md5B40BAC) to 94% MD after B13-IVP5 Md5B40BAC challenge. In the MHC-B21-resistant line MD virulence fluctuated from 28% at B21-IVP1 Md5B40BAC to a high of 65% in B21-IVP2 Md5B40BAC back to a low of 23% in B21-IVP5 Md5B40BAC-challenged chicks. Although the B21-IVP5 Md5B40BAC isolates were relatively mild in the MHC-B21 chicken line (56% MDV), they were highly virulent in the MHC-B13 line (100% MDV). From this series of experiments it would appear that MDV evolution toward greater virulence occurs in both susceptible and resistant MHC haplotypes, but the resulting increase in pathogenicity is constrained by the resistant MHC haplotype.

MHC class I target recognition, immunophenotypes and proteomic profiles of natural killer cells within the spleens of day-14 chick embryos.

Chicken natural killer (NK) cells are not well defined, so little is known about the molecular interactions controlling their activity. At day 14 of embryonic development, chick spleens are a rich source of T-cell-free CD8αα(+), CD3(-) cells with natural killing activity. Cell-mediated cytotoxicity assays revealed complex NK cell discrimination of MHC class I, suggesting the presence of multiple NK cell receptors. Immunophenotyping of freshly isolated and recombinant chicken interleukin-2-stimulated d14E CD8αα(+) CD3(-) splenocytes provided further evidence for population heterogeneity. Complex patterns of expression were found for CD8α, chB6 (Bu-1), CD1-1, CD56 (NCAM), KUL01, CD5, and CD44. Mass spectrometry-based proteomics revealed an array of NK cell proteins, including the NKR2B4 receptor. DAVID and KEGG analyses and additional immunophenotyping revealed NK cell activation pathways and evidence for monocytes within the splenocyte cultures. This study provides an underpinning for further investigation into the specificity and function of NK cells in birds.

Serial transfer of a transplantable tumor: implications for Marek's vaccine mechanisms.

The mechanism of Marek's disease (MD) vaccination to prevent the lymphoproliferative disease in chickens is not well understood. It is generally recognized that vaccination prevents disease, including the induction of T-cell tumors, but it does not prevent the pathogenic virus from infecting and replicating in the vaccinated host, nor does it prevent bird to bird spread of the oncogenic virus. The stage at which the vaccinated immune system intervenes in the process from infection to the induction of tumors remains obscure. Using a transplantable tumor induced by the Md5 strain of MD virus (MDV), we show that CVI988 vaccination does not prevent the induction of transplantable tumors in the 15I(5) x 7(1) chicken line. A monoclonal tumor with a V beta 1 T-cell receptor spectratype of 207 base pairs was used to follow the transplantable tumor in serial passages in vivo. This transplantable tumor could be passed in vaccinated birds. The length of time between vaccination and challenge (5 to 12 days) had little or no influence on the ability to transfer the tumor. There was variability in the manifestation of the disease produced by the transplanted tumor. Some chickens presented as normal but were still capable of transmitting the transplanted tumor to newly vaccinated recipients via their blood. This indicates that some chickens can control, but not eliminate, the tumor. The variables inducing health or disease in the challenged chickens remain obscure, but environmental or other factors likely depress the immune system allowing the tumor to overwhelm the immune system.

Down-regulation of MHC class I by the Marek's disease virus (MDV) UL49.5 gene product mildly affects virulence in a haplotype-specific fashion.

Marek's disease is a devastating neoplastic disease of chickens caused by Marek's disease virus (MDV). MDV down-regulates surface expression of MHC class I molecules, although the mechanism has remained elusive. MDV harbors a UL49.5 homolog that has been shown to down-regulate MHC class I expression in other Varicelloviruses. Using in vitro assays, we showed that MDV pUL49.5 down-regulates MHC class I directly and identified its cytoplasmic tail as essential for this function. In vivo, viruses lacking the cytoplasmic tail of pUL49.5 showed no differences in MD pathogenesis compared to revertant viruses in highly susceptible chickens of the B(19)B(19) MHC class I haplotype, while there was a mild reduction in pathogenic potential of the deletion viruses in chickens more resistant to MD pathogenesis (MHC:B(21)B(21)). We concluded that the pathogenic effect of MHC class I down-regulation mediated by pUL49.5 is small because virus immune evasion possibly requires more than one viral protein.

Major histocompatibility complex and background genes in chickens influence susceptibility to high pathogenicity avian influenza virus.

The chicken's major histocompatibility complex (MHC) haplotype has profound influence on the resistance or susceptibility to certain pathogens. For example, the B21 MHC haplotype confers resistance to Marek's disease (MD). However, non-MHC genes are also important in disease resistance. For example, lines 6 and 7 both express the B2 MHC haplotype, but differ in non-MHC genes. Line 6, but not line 7, is highly resistant to tumors induced by the Marek's disease herpesviruses and avian leukosis retroviruses. Recently, survival in the field by Thai indigenous chickens to H5N1 high-pathogenicity avian influenza (HPAI) outbreaks was attributed to the B21 MHC haplotype, whereas the B13 MHC haplotype was associated with high mortality in the field. To determine the influence of the MHC haplotype on HPAI resistance, a series of MHC congenic white leghorn chicken lines (B2, B12, B13, B19, and B21) and lines with different background genes but with the same B2 MHC haplotype (Line 63 and 7(1)) were intranasally challenged with low dose (10 mean chicken lethal doses) of reverse-genetics-derived rg-A/chicken/Indonesia/7/2003 (H5N1) HPAI virus. None of the lines were completely resistant to lethal effects of the challenge, as evidenced by mortality rates ranging from 40% to 100%. The B21 line had mortality of 40% and 70%, and the B13 line had mortality of 60% and 100% in two separate trials. In addition, the mean death times varied greatly between groups, ranging from 3.7 to 6.9 days, suggesting differences in pathogenesis. The data show that the MHC has some influence on resistance to AI, but less than previously proposed, and non-MHC background genes may have a bigger influence on resistance than the MHC.

Marek's disease viruses lacking either R-LORF10 or LORF4 have altered virulence in chickens.

The Marek's disease virus (MDV, Gallid herpesvirus 2) genome encodes approximately 110 open reading frames (ORFs). Many of these ORFs are annotated based purely on homology to other herpesvirus genes, thus, direct experiments are needed to verify the gene products, especially the hypothetical or MDV-specific ORFs, and characterize their biological function, particularly with respect to pathogenicity in chickens. Previously, a comprehensive two-hybrid assay screen revealed nine specific chicken-MDV protein-protein interactions. In order to characterize the role of hypothetical MDV proteins R-LORF10 and LORF4, which were shown to interact with major histocompatibility complex (MHC) class II beta chain and Ii (invariant or gamma) chain, respectively, recombinant MDVs derived from virulent MDV-BAC clone rMd5-B40 were generated. Recombinant MDV rMd5DeltaR-LORF10 lacked part of the promoter and the first 17 amino acids in both copies of R-LORF10, and rMd5mLORF4 had point mutations in LORF4 that disrupted the start codon and introduced a premature stop codon without altering the amino acid sequence of overlapping ORF UL1, which encodes glycoprotein L (gL). Mutations in either R-LORF10 or LORF4 neither prevent MDV reconstitution from modified MDV-BACs nor significantly alter virus growth rate in vitro. However, MDV generated from rMd5DeltaR-LORF10 had reduced virulence compared to the parental MDV. Surprisingly, MDV with the LORF4 mutations had significantly higher overall MD incidence as measured by mortality, tumor production, and MD symptoms in infected chickens. These results indicate R-LORF10 and LORF4 encode real products, and are involved in MDV virulence although their mechanisms, especially with respect to modulation of MHC class II cell surface expression, are not clearly understood.

Cloning and functional characterization of chicken stem cell antigen 2.

Stem cell antigen 2 (SCA2) is a Ly6 family member whose function is largely unknown. To characterize biological properties and tissue distribution of chicken SCA2, SCA2 was expressed in E. coli, purified, and a polyclonal antibody developed. Utilizing the polyclonal antibody, SCA2 is a 13 kDa cell surface protein anchored by a glycosyl-phosphatidylinositol (GPI) moiety. SCA2 is expressed in connective tissues of thymus and bursa based on immunohistochemistry, immunoprecipitation, and western blots. In bursal follicles, SCA2 is specifically expressed on the cortical-medullary epithelial cells (CMEC) surrounded by MHC class II presenting cells. Expression profiles of bursal cells induced by contact with SCA2-expressing cells shows down-regulation of numerous genes including CD79B, B cell linker (BLNK), spleen tyrosine kinase (SYK), and gamma 2-phospholipase C (PLCG2) that are involved in the B cell receptor (BCR) and immune response signaling pathways. These results suggest chicken SCA2 plays a role in regulating B lymphocytes.

Chromosomal integration of an avian oncogenic herpesvirus reveals telomeric preferences and evidence for lymphoma clonality.

BACKGROUND: Herpesviruses are a major health concern for numerous organisms, including humans, causing both acute and chronic infections recurrent over an individual's lifespan. Marek's disease virus (MDV) is a highly contagious herpesvirus which causes a neoplastic condition in chicken populations. Several vertebrate-infecting herpesviruses have been shown to exist in an integrated state during latent periods of infection. However the status of MDV during latency has been a topic of debate. RESULTS: Here we employed high-resolution multi-color fluorescence in situ hybridization (FISH) to show integration of MDV at the telomeres of chicken chromosomes. Cytogenomic mapping of the chromosomal integrations allowed us to examine the clonal relationships among lymphomas within individuals, whereas analysis of tumors from multiple individuals indicated the potential for chromosomal preferences. CONCLUSIONS: Our data highlight that substantive genome-level interactions between the virus and host exist, and merit consideration for their potential impact and role in key aspects of herpesvirus pathobiology including infection, latency, cellular transformation, latency-breaks and viral evolution.

Mass spectral data for 64 eluted peptides and structural modeling define peptide binding preferences for class I alleles in two chicken MHC-B haplotypes associated with opposite responses to Marek's disease.

In the chicken, resistance to lymphomas that form following infection with oncogenic strains of Marek's herpesvirus is strongly linked to the major histocompatibility complex (MHC)-B complex. MHC-B21 haplotype is associated with lower tumor-related mortality compared to other haplotypes including MHC-B13. The single, dominantly expressed class I gene (BF2) is postulated as responsible for the MHC-B haplotype association. We used mass spectrometry to identify peptides and structural modeling to define the peptide binding preferences of BF2 2101 and BF2 1301 proteins. Endogenous peptides (8-12 residues long) were eluted from affinity-purified BF2 2101 and BF2 1301 proteins obtained from transduced cDNA expressed in RP9 cells, hence expressed in the presence of heterologous TAP. Sequences of individual peptides were identified by mass spectrometry. BF2 2101 peptides appear to be tethered at the binding groove margins with longer peptides arching out but selected by preferred residues at positions P3, P5, and P8: X-X-[AVILFP]-X((1-5))-[AVLFWP]-X((2-3))-[VILFM]. BF2 1301 peptides appear selected for residues at P2, P3, P5, and P8: X-[DE]-[AVILFW]-X((1-2))-[DE]-X-X-[ED]-X((0-4)). Some longer BF2 1301 peptides likely also arch out, but others are apparently accommodated by repositioning of Arg83 so that peptides extend beyond the last preferred residue at P8. Comparisons of these peptides with earlier peptides derived in the presence of homologous TAP transport revealed the same side chain preferences. Scanning of Marek's and other viral proteins with the BF2 2101 motif identified many matches, as did the control human leukocyte antigen A 0201 motif. The BF2 1301 motif is more restricting suggesting that this allele may confer a selective advantage only in infections with a subset of viral pathogens.

Marek's disease virus up-regulates major histocompatibility complex class II cell surface expression in infected cells.

Many herpesviruses modulate major histocompatibility complex (MHC) expression on the cell surface as an immune evasion mechanism. We report here that Marek's disease virus (MDV), a lymphotrophic avian alphaherpesvirus, up-regulates MHC class II cell surface expression in infected cells, contrary to all other herpesviruses examined to date. This MDV-induced class II up-regulation was detected both in vitro and in vivo. This effect was not solely an indirect effect of interferon, which is a highly potent natural inducer of MHC class II expression, since MHC class II up-regulation in cultured primary fibroblast cells was confined to the infected cells only. MHC class II up-regulation was also observed in infected cells of the bursa of Fabricius during the lytic phase of MDV infection in birds and upon reactivation of MDV from latency in an MDV-transformed cell line. As MDV is a strictly cell-associated virus and requires activated T cells for its life cycle, this up-regulation of MHC class II in infected cells may contribute to virus spread within the infected host by increasing the chance of contact between productively infected cells and susceptible activated T cells.

At least one YMHCI molecule in the chicken is alloimmunogenic and dynamically expressed on spleen cells during development.

Transcriptionally active, MHC class I (MHCI) loci are located in two separate polymorphic genomic regions in the chicken called B and Y. The YMHCI gene sequences encode molecules with uncommon substitutions in the antigen-binding region indicating that YMHCI molecules are likely unique and may bind a specialized form of antigen distinct from that of other antigen-binding MHCI molecules. To learn whether YMHCI gene expression results in the production of alloantigens at the cell surface, we immunized 15I(5) x 7(2) : chickens using syngeneic RP9 cells expressing transduced YF1w*7.1, a potentially alloimmunogenic YMHCI allele from the Y7 haplotype present in line C. The resulting antisera show that YF1w*7.1 MHCI molecules are immunogenic and expressed on the surfaces of cells in blood and spleen of line C chickens. Virtually all CD3+, CD4+, and CD8+ cells circulating in line C blood are positive, as are BU1+ cells. The YF1w*7.1 MHCI allele is dynamically expressed at levels comparable to but transcriptionally independent of classical BMHCI on erythrocytes, lymphocytes, granulocytes, monocytes, and thrombocytes within the spleen pre- and post-hatching. The antisera react with cells from two among four haplotypes segregating in closed populations of lines N and P. YMHCI shares features associated with both classical and non-classical MHCI. It is becoming increasingly likely that YMHCI has a fundamental role in avian immunity and thereby needs to be included in the growing spectrum of functionally active, diverse MHCI molecules no longer adequately described by the classical/non-classical dichotomy.