https://orcid.org/0000-0003-0324-162X
Figures
Abstract
Marek’s disease virus (MDV), an alphaherpesvirus, causes severe immunosuppression and T cell lymphomas in chickens, known as Marek’s disease (MD), an economically important poultry disease primarily controlled by vaccination. Importantly, it also serves as a comparative model for studying herpesvirus-induced tumor formation in humans. MDV encodes more than 100 genes, most of which have unknown functions. MDV LORF1 is unique to serotype I MDV (MDV-1), lacking homologs in other herpesviruses, and has not been explored yet. To this end, an infectious bacterial artificial chromosome (BAC) harboring the complete genome of the MDV-1 very virulent strain Md5 was generated, and the rescued rMd5 maintained biological properties similar to the parental virus both in vitro and in vivo. Subsequently, rMd5ΔLORF1, a recombinant Md5 virus deficient in pLORF1 expression, was generated by a frameshift mutation in the LORF1 gene. Chickens infected with rMd5ΔLORF1 exhibited a lower mortality rate and delayed bursal atrophy than those infected with the parental rMd5 and the revertant virus (rMd5-reLORF1). Consistently, viral loads of rMd5ΔLORF1 were obviously lower than those of rMd5 or rMd5-reLORF1 in the bursa, but not in the spleen. Importantly, we found that pLORF1 deficiency impairs viral replication in bursal B cells. Furthermore, we showed that pLORF1 associated with the cellular membrane, interacted with MDV structural proteins, and exhibited punctate colocalization with tegument or capsid proteins in the cytoplasm. Taken together, this study demonstrates for the first time that the MDV-1 unique gene LORF1 is involved in MDV-induced bursal atrophy but not in tumor formation.
Author summary
Marek’s disease virus (MDV) is an extensively studied alphaherpesvirus since it is not only a causative agent of Marek’s disease (MD), a highly contagious poultry neoplastic disease posing a significant economic impact on the poultry industry worldwide, but also a comparative model for studying human oncogenic viruses. Although MD has been effectively controlled by massive vaccination since 1969, the continuous emergence of higher virulent strains creates challenges for preventing future outbreaks due to vaccine failure. Therefore, the characterization of unknown genes related to MDV-induced tumor formation and/or immune suppression is of great importance. In this study, we showed for the first time that LORF1, a unique gene of serotype 1 MDV, appears to affect viral replication within the bursa, consequently influencing bursal atrophy. Our findings reveal an essential role of LORF1 in immune suppression, suggesting its potential as a target gene for the development of an MDV gene deletion vaccine.
Citation: Bao C, Chu J, Gao Q, Yang S, Gao X, Chen W, et al. (2025) Marek’s disease virus-1 unique gene LORF1 is involved in viral replication and MDV-1/Md5-induced atrophy of the bursa of Fabricius. PLoS Pathog 21(2): e1012891. https://doi.org/10.1371/journal.ppat.1012891
Editor: Wolfram Brune, Leibniz Institute of Virology (LIV), GERMANY
Received: July 19, 2024; Accepted: January 7, 2025; Published: February 3, 2025
Copyright: © 2025 Bao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: This work was supported by the National Natural Science Foundation of China (31472218 to YQ), the Fundamental Research Funds for the Central Universities (KYCXJC2024005 to YQ), the Natural Science Foundation of Jiangsu Province (BK20140711 to YQ), and the National Natural Science Foundation of China (32202799 to XL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Marek’s disease virus (MDV) is a highly pathogenic, immunosuppressive, and oncogenic alphaherpesvirus primarily affecting chickens [1]. MDV belongs to the genus Mardivirus, subfamily Alphaherpesvirinae, and family Herpesviridae. The genus Mardivirus comprises three closely related but distinct species: MDV-1 (Gallid herpesvirus 2, GaHV-2), GaHV-3 (previously MDV-2), and turkey herpesvirus 1 (HVT; Meleagrid herpesvirus 1, MeHV-1; previously MDV-3). The sequence similarity of viral proteins of the three species ranges from 50% to 80% [2–4]. However, only MDV-1 causes Marek’s disease (MD) in chickens, which is not only economically important to the global poultry industry but also an excellent natural T cell lymphoma model to improve our understanding of herpesvirus-induced tumorigenesis in humans [5]. For example, MDV-induced lymphomas share many similarities with lymphoid tumors associated with human herpesviruses, such as Epstein-Barr virus (EBV) [6]. Despite the success of vaccines in reducing losses from MD over the past four decades, the virus has evolved to be more virulent in vaccinated chickens, posing a threat to the effectiveness of the vaccine [7]. Three different pathotypes of the virus have been identified based on their vaccine escape abilities: virulent (v), very virulent (vv), and very virulent plus (vv+) [8]. A better understanding of the pathogenicity and oncogenicity of MDV is essential not only to clarify virus-associated pathogenesis but also to develop more effective vaccines to combat the infection.
Bacterial artificial chromosome (BAC) clones allow rapid manipulation of viral genomes for characterizing gene function and generating recombinant vaccines. In a seminal study, the complete genome of an attenuated strain of MDV vv+ 584A virus (584Ap80C) was established as a BAC clone [9]. The BAC sequence was inserted into the US2 locus of the MDV genome, and the recombinant virus was enriched with a selection media containing mycophenolic acid, xanthine, and hypoxanthine [9]. BAC clones from MDV pathogenic and vaccine strains were later extensively developed following the same principle [10–18]. Since the beginning of the 21st century, multiple key genes involved in MDV-induced pathogenesis, tumorigenesis, and immune suppression have been identified through genetically specific deletion and insertion modifications to MDV genes using these MDV BAC clones [19–27]. Meanwhile, recombinant MDV vaccine candidates deficient in important virulent genes, such as Meq, have been explored and shown vaccine potential [28,29]. MDV-1 encodes at least 100 gene products, with the majority situated in the unique long (UL) and unique short (US) regions of the viral genome [30]. These genes exhibit homology to those found in herpes simplex virus (HSV-1) and are essential for various viral processes such as genomic DNA replication, gene expression, virus assembly, and morphogenesis [5,31]. For instance, genes like glycoprotein B (gB) and gM in the UL region are critical for MDV replication in vitro [9,32]. UL28 and UL33 are crucial for MDV packaging and maturation [33]. Genes in the US region encode important viral envelope glycoproteins (gD, gI, and gE), and the US3 protein kinase is involved in DNA replication and gene expression [34–38]. Several unique genes, such as Meq, vIL8, vTR, pp38, and vLIP, identified within the repeat long (RL) regions and the UL region, are known to play significant roles in MDV pathogenesis and/or oncogenesis [19,21,39–46]. However, many other open reading frames (ORFs) unique to MDV or to Mardivirus viruses in general have not been thoroughly studied. Notably, there are 16 unique ORFs in the RL region, 12 in the UL region, and 4 in the US region [31,47]. These unique genes, absent in GaHV-3, HVT, or other herpesviruses, likely contribute to the distinct biological properties of these viruses. Despite this, many of these ORFs remain uncharacterized, with limited homology to known genes or sequence motifs. Some ORFs overlap with other MDV genes, and mutations affecting start or stop codons can help elucidate their specific functions without altering the protein sequence of the overlapping genes. This necessitates a technological platform for precise manipulation of the viral genome to better elucidate their functions.
LORF1, the first ORF of the UL region of the MDV-1 genome, was first identified as ORF-2, encoding a putative protein consisting of 333 amino acids [31,48], or MDV009 due to its presence in MDV-1, but not in MDV-2 or HVT [30]. Two unique peptides of pLORF1 were identified through proteomic analysis of Md5-infected chicken embryo fibroblasts (CEFs) cells [49]. Expression of LORF1 mRNA was detected in an MDV-transformed tumor cell line [50]. However, its role in MDV pathogenesis remains unknown [47]. In this study, we demonstrate that pLORF1 is involved in MDV-1-induced immune suppression but not in tumor formation.
Results
Construction of a BAC containing the complete MDV-1 genome
In order to explore the role of unknown genes of MDV-1, an MDV-BAC clone containing the full-length MDV-1 Md5 genome was generated, as illustrated in Fig 1. The transfer vector pBlue-US2-BAC-RFP carries a homologous recombination cassette consisting of homology arms (US2A and US2B), a loxP-flanked partial sequence of the pBeloBAC11 plasmid, and a selection marker expression cassette (RFP and puromycin) (Fig 1B). The pBeloBAC11 contains the CmR gene, the Ori2 replicon, and the replication and partition genes (repE, sopA, sopB, and sopC). This transfer vector directs the insertion of BAC elements into the US2 gene of MDV (Fig 1A) as previously reported [9]. CEFs were transfected with the linear pBlue-US2-BAC-RFP vector and subsequently infected with the Md5 virus. Instead of using selection medium to enrich recombinant viruses, a common method used in constructing MDV-BAC [9–18], we utilized the red fluorescent protein (RFP) to visualize recombinant virus-formed plaques for purification. Transfected/infected cells were trypsinized and serially diluted onto fresh CEFs in 96-well plates. Wells containing a single RFP-positive plaque were selected to repeat the above steps. After six rounds of plaque purification, the propagation of the recombinant Md5-BAC-RFP virus (Fig 1C) was observed with the aid of a fluorescence microscope, and the images revealed that all plaques fluoresced in red (Fig 1D). The circular viral DNAs (Fig 1E) from these cultures were transformed into E. coli DH10B using electroporation. Chloramphenicol-resistant colonies were screened using restriction fragment length polymorphism (RFLP) analysis. Two independently derived BAC clones, named pMd5/14-4 and pMd5/14-20, were confirmed by restriction enzyme digestion using EcoRI, BamHI, HindIII, and SacI, respectively. No noticeable deletions, insertions, or rearrangements in restriction patterns were identified in the pMd5 DNA by comparing electrophoretic profiles with those predicted by the software (Fig 2A and S1A Fig). The asterisks indicate the fragments generated due to the insertion of the transfer vector into the Md5 genome.
(A) Schematic of the linear MDV-1 Md5 genome and a portion of the unique short region (US) containing US10 to US6 ORFs and the location of homologous arms (horizontal dashed line). (B) A linearized transfer vector, pBlue-US2-BAC-RFP, includes homologous arms (US2A and US2B), a loxP-flanked partial sequence of the pBeloBAC11 plasmid, and a selection marker expression cassette (RFP and puromycin). (C) CEFs were transfected with linearized pBlue-US2-BAC-RFP, followed by MDV-1 Md5 virus infection. Through classical homologous recombination, the transfer vector was introduced into the MDV-1 genome between SORF3 and US3 to yield a recombinant MDV-1, Md5-BAC-RFP. (D) Microscopic observation of red fluorescent plaques of Md5-BAC-RFP obtained by plaque purification in CEFs. (E) Md5-BAC-RFP viral DNA was isolated from infected CEFs and electroporated into E. coli DH10B cells to generate infectious pMd5 clones. (F and G) CEFs were transfected with pMd5 DNA with or without the Cre recombinase expression plasmid and produced recombinant virus rMd5-BAC-RFP (F) or rMd5 (G). The blue arrows indicate the locations of primers used to confirm the removal of the BAC sequence.
(A) RFLP analysis of BAC DNA. Electrophoresis of pMd5 DNAs (clones 14–4 and 14–20) that were cleaved with EcoRI, BamHI, HindIII, or SacI endonucleases. The bands that indicate the insertion of the transfer vector were marked with asterisks. (B) The microscopic images of viral plaques of Md5, rMd5/14-4, and rMd5/14-20 in CEFs at 5 dpi. (C) The growth curve of Md5, rMd5/14-4, or rMd5/14-20 was performed as described in “Materials and Methods.” Each value represents the mean of two independent duplicates. (D-G) One-day-old SPF chickens were randomly assigned to receive intraperitoneal injections of either Md5 or rMd5 at a dose of 1,000 PFU, or DMEM as a control. The chickens were kept in isolators for 54 days together with contact chickens housed in the same isolators as either the Md5 or rMd5 group. (D) Survival curves of chickens inoculated with the indicated viruses and the control group. (E) The viral load in contacted chickens. The levels of the MDV VP22 and ICP4 genes were measured by PCR using DNA purified from the spleen tissues of mock-treated or contact chickens from the Md5 or rMd5 groups at 54 dpi. (F and G) Liver (F) and heart (G) tissues from chickens at 42 dpi in each group were stained with H&E. The black boxes indicate large populations of lymphoid tumor cells scattered in the Md5- or rMd5-infected tissues. Scale bar: 50 μm.
To rescue the recombinant rMd5-BAC-RFP virus (Fig 1F) from pMd5 BAC (Fig 1E), CEFs were transfected with pMd5/14-4 or pMd5/14-20 DNA, and red fluorescent plaques were visualized at 5 days post-transfection (S1B Fig). To remove the BAC vector flanked by loxP sites from the pMd5 genome to obtain a minimally modified MDV, rMd5 (Fig 1G), CEFs were co-transfected with pMd5 DNA and a Cre expression plasmid. Non-fluorescent BAC-excised rMd5 viruses were plaque-purified and confirmed by PCR amplification of the DNA from rMd5-infected CEFs with primers shown in Fig 1F (blue arrows). The results showed that the US2 gene junctions, RFP, and sopA were exclusively detected in rMd5-BAC-RFP, but not in rMd5 samples, while the VP22 remained intact in all viruses (S1C Fig). The recombinant viruses, rMd5/14-4 and rMd5/14-20, induced typical MDV cytopathic effects, similar to those of the wild-type Md5 virus (Fig 2B). In addition, the growth kinetics of rMd5/14-4 and rMd5/14-20 closely resembled that of the parental Md5 on CEFs (Fig 2C). Since both rMd5/14-4 and rMd5/14-20 displayed indistinguishable properties in vitro, clone 14–20 was used for further experiments.
Characterization of BAC-derived MDV-1 in vivo
The virulence of rMd5 was determined in one-day-old SPF chickens over a 54-day period, with Md5 as a control. MDV infection caused clinical symptoms or death, which were monitored and showed that chickens started to die from 21 dpi, and the median survival time was 41 dpi in both rMd5- and Md5-infected groups (Fig 2D). In addition, to examine the horizontal transmission of rMd5, the levels of VP22 and ICP4 genes in the spleens of contact chickens were analyzed by PCR at the end of the experiment. The results showed that VP22 and ICP4 were detected in all contact chickens in both Md5 and rMd5-infected groups, indicating that the rMd5 virus maintains the ability to spread horizontally from infected chickens to contact chickens (Fig 2E).
Since MDV infection induces immunosuppression and causes abnormalities in lymphoid organs in chickens, chicken bursas and spleens were examined at 28 dpi, and those organs showed that both rMd5 and Md5 infections induced bursal atrophy and spleen enlargement (S1D Fig). In addition, MDV-induced lymphoma formation was also observed in the livers of both infection groups at 42 dpi (S1E Fig). Moreover, histological examination of dissected liver and heart tissues showed typical lymphocyte infiltration in these tissues of both Md5 and rMd5-infected chickens (Fig 2F and 2G, and S1F Fig), as well as in liver tissues of contact chickens (S1G Fig). These results suggest that the BAC-derived rMd5 virus maintains the ability to induce MD in chickens, comparable to the parental Md5 virus.
Construction and characterization of recombinant Md5 viruses in vitro
MDV LORF1 is unique to MDV-1 and has no homolog in other herpesviruses [30]. To date, its function in MDV pathogenesis remains unknown [47]. According to the MDV genome structure, the LORF1 gene completely overlaps with R-LORF14 and R-LORF13, partially overlaps with R-LORF12, and is adjacent to LORF2 (Fig 3A). Therefore, rather than creating a full-length deletion, a LORF1 frameshift mutant, rMd5ΔLORF1, was constructed by deleting AA at base positions 7 and 8 using the Red-mediated recombination system [51], which generated in-frame 14 premature stop codons, among which one locates at the second position after the translational start site (Fig 3A). Meanwhile, a revertant virus, rMd5-reLORF1, was also generated using Md5-BAC (Fig 3A). The RFLP analysis showed that both pMd5ΔLORF1 and pMd5-reLORF1 displayed the expected HindIII-digested DNA profiles (Fig 3B). Since the 2-bp deletion in the LORF1 gene cannot be differentiated only based on DNA fragment size, PCR products targeting the deletion region of pMd5ΔLORF1 and pMd5-reLORF1 were sequenced and showed that the LORF1 gene was precisely edited in each clone (Fig 3C). rMd5ΔLORF1 and rMd5-reLORF1 were rescued as above. To confirm the effect of the mutation on LORF1 expression, the C-terminal region of pLORF1 (174–333 amino acids, black arrow in Fig 3A) was expressed as an antigen to produce antisera against pLORF1 in mice. Confocal imaging revealed that the anti-pLORF1 antibody detected punctate localization of pLORF1 in the cytoplasm of cells infected with rMd5 and rMd5-reLORF1, which was absent in cells infected with rMd5ΔLORF1 (Fig 3D). Finally, the growth curve of the rMd5ΔLORF1 virus was comparable to the parental and revertant viruses (Fig 3E), suggesting that silencing the expression of the LORF1 gene had no effect on Md5 replication in CEFs.
(A) Schematic diagrams of the genome structure of MDV Md5 and the mutant viruses. Line 1: the wild-type MDV Md5 genome; Line 2: a segment of the Md5 genome that encompasses the R-LORF12 to LORF2 ORFs; Line 3: the rMd5ΔLORF1 virus with newly created stop codons (asterisks) due to a 2-bp deletion in the LORF1 gene; Line 4: the rMd5-reLORF1 virus with restored wild-type LORF1 ORF by re-introduction of 2 bp in the mutant rMd5ΔLORF1. The C-terminal region of pLORF1 (174–333 aa) was expressed as an antigen to produce anti-pLORF1 antibodies (black arrow). (B) RFLP analysis of HindIII-digested BAC DNAs from pMd5, pMd5ΔLORF1, or pMd5-reLORF1. (C) PCR amplification and sequencing results of the LORF1 gene in pMd5ΔLORF1 or -reLORF1. (D) The subcellular localization of pLORF1. CEFs were infected with rMd5, rMd5ΔLORF1, or rMd5-reLORF1 for 3 days, followed by fixation and staining with anti-pLORF1 antibody and Alexa 555-conjugated goat anti-mouse IgG antibody (red). The nuclei were stained with DAPI (blue). Original magnification: ×600. (E) The growth curve of rMd5, rMd5ΔLORF1, or rMd5-reLORF1. Each value represents the mean of two independent duplicates.
Previously, studies demonstrated that the deletion of Meq completely disabled Md5-induced tumor formation [39]. Therefore, a full-length Meq-deleted virus, rMd5ΔMeq, was simultaneously constructed as a control (S2A and S2B Fig) and confirmed by RFLP analysis and PCR amplification (S2C and S2D Fig). Consistent with previous findings, the deletion of Meq did not affect Md5 replication in CEFs (S2E Fig).
Characterization of recombinant Md5 viruses in vivo
To investigate the role of pLORF1 in MDV pathogenesis in vivo, one-day-old SPF chickens were intraperitoneally inoculated with 1,000 PFU of the parental virus (rMd5), the mutant virus (rMd5ΔLORF1), the revertant virus (rMd5-reLORF1), or the Meq-deleted virus (rMd5ΔMeq), respectively, along with DMEM as a negative control. The MD symptoms and survival of infected chickens were monitored for 60 days. Compared to the rMd5 and rMd5-reLORF1 groups, the rMd5ΔLORF1 group showed delayed progression of MD and lower mortality rates. The mortality rate of the rMd5ΔLORF1 group was 55% at the end of the experiment, while it was 90% for the rMd5 group and 95% for the rMd5-reLORF1 group (Fig 4A). There were no MDV-associated deaths in the rMd5ΔMeq group (Fig 4A), as previously reported [39]. In addition, 82% (9/11 chickens) of the chickens that died due to rMd5ΔLORF1 infection were clustered between 35 and 60 dpi (Fig 4A, marked by two red dotted lines). Interestingly, we found that rMd5ΔLORF1-infected chickens that died during this period had significantly higher body weights than those in the rMd5 and rMd5-reLORF1 groups (Fig 4B). Meanwhile, bursal atrophy in rMd5ΔLORF1-infected chickens was not as severe as that in the case of the rMd5 and rMd5-reLORF1 viruses (S3A Fig).
The animal experiment was performed as described in “Materials and Methods.” (A) Survival curves of chickens inoculated with the indicated viruses and the control group. The time interval indicated by the two red dotted lines represents when 82% (9/11 chickens) of rMd5ΔLORF1-infected chickens died. (B) The body weight of dead chickens in each group from 35 dpi to 60 dpi. (C) The bursa to body weight ratio of chickens from each group at the indicated time points. Results represent the mean value, with error bars indicating the standard error of the mean. (D) The level of the MDV VP22 gene was measured by PCR using DNA purified from bursa and spleen samples of chickens from each group at the indicated time points.
To confirm the aforementioned findings, another flock of one-day-old chickens was subjected to the same treatments (DMEM, rMd5, rMd5ΔLORF1, rMd5-reLORF1, and rMd5ΔMeq) and randomly selected for euthanasia at 7, 14, 21, 28, 35, or 42 dpi, respectively. During the observation period, chickens from the rMd5ΔLORF1 group exhibited progressively higher body weights compared to those in the rMd5, rMd5-reLORF1, and rMd5ΔMeq groups until 35 dpi (S3B Fig and S1 Table). To evaluate the effect of Md5-induced bursal atrophy, bursas were dissected, photographed (S3C Fig), and weighed (S1 Table). To rule out the effect of absolute weight of body or bursa due to exceptional individuals [52], the bursa/body weight ratio was calculated and shown to be continuously higher in chickens infected with rMd5ΔLORF1 since 14 dpi than in the rMd5-reLORF1 and rMd5ΔMeq groups (Fig 4C). The viral loads in the bursa and spleen tissues were analyzed by measuring the level of genomic VP22 and showed that it was substantially lower in the rMd5ΔLORF1-infected bursa samples than in rMd5- or rMd5-reLORF1-infected samples at 7, 28, and 35 dpi, but not at 42 dpi (Fig 4D), while it remained unchanged in the spleen (Fig 4D). To examine the tumorigenesis of recombinant viruses, histological assessment of liver tissues showed that liver lymphocyte infiltration in rMd5ΔLORF1-infected chickens was as severe as in the rMd5 and rMd5-reLORF1 groups at 35 dpi (S4 Fig).
Histopathological analysis of bursal tissues infected by recombinant viruses
To further analyze the effect of pLORF1 on viral replication in the bursa, H&E staining was performed on bursal tissues collected at 7 and 42 dpi. As shown in Fig 5A–5H, bursal tissues from the control group showed a well-preserved architectural structure characterized by folds and follicles, along with minimum inter-follicular connective tissue septa (indicated by “CTS”). The follicles comprised the medulla and cortex, separated by a single-celled layer of endodermal epithelium (marked as “M,” “C,” and “CME”). The medulla of the follicle contained loosely dispersed small lymphocytes, while the cortex was populated with large lymphocytes tightly packed with cells from the plasmacyte lineage. In contrast, bursal tissues from the rMd5 or rMd5-reLORF1 group displayed infiltration of reticular cells at 7 dpi (Fig 5A and 5B), and showed severely atrophied follicles with a noticeable increase in the thickness of the endodermal epithelium or fuzzy demarcated cortico-medullary regions, accompanied by lymphocytic depletion and necrosis in both the medulla and cortex at 42 dpi (Fig 5E and 5F). Reticular cells and heterophile infiltration, as well as severe inter-follicular fibroplasia, were also observed. Conversely, bursal tissues from rMd5ΔLORF1-infected chickens exhibited a normal follicular structure at 7 dpi (Fig 5A and 5B), and showed moderate follicular atrophy with lymphocytic infiltration and thickened inter-follicular connective tissue at 42 dpi (Fig 5E and 5F).
The bursa tissues from chickens that were mock-treated or inoculated with 1,000 PFU of rMd5, rMd5ΔLORF1, or rMd5-reLORF1, and collected at 7 dpi (A-D) and 42 dpi (E-H). (A and E) Low-magnification images of HE-stained bursa tissues. (B and F) A higher magnification of the areas indicated by red boxes in A or E. (C and G) Low-magnification images of immunohistochemistry of bursa tissues. Bursal sections stained with anti-VP22 antibody and HRP-conjugated goat anti-mouse IgG antibody were visualized using DAB (brown), while the nuclei were stained with hematoxylin (blue). (D and H) Higher magnifications of the areas indicated by the red boxes in panels C or G. (I) Bursal sections at 7 dpi were stained with anti-Bu-1 and Alexa 488-conjugated goat anti-mouse IgG antibodies (green), followed by staining with anti-VP22 and Alexa 555-conjugated goat anti-mouse IgG antibodies (red). The nuclei were stained with DAPI (blue). CTS, Connective Tissue Septum; CME, Cortico-Medullary Epithelium; C, Cortex; M, Medulla. Scale bar: 1 mm (A, C, E, and G), 20 μm (B, D, F, and H).
To investigate the lytic replication of MDV within the bursa of Fabricius, an immunohistochemistry assay was performed on bursal tissues infected with recombinant viruses by staining with an anti-VP22 antibody. The results showed that rMd5ΔLORF1-infected bursal tissues were negative following VP22 staining at 7dpi (Fig 5C and 5D) and showed a small amount of positive staining at 42 dpi (Fig 5G and 5H). Given that the bursa of Fabricius is where B cells develop and mature, to further examine whether pLORF1 affects MDV replication in B cells, bursal sections were immunostained with anti-Bu-1 (a B cell surface marker) and anti-VP22 antibodies. Confocal imaging revealed that VP22 was primarily colocalized with Bu-1 in rMd5-infected samples, but VP22 staining was negative in rMd5ΔLORF1-infected samples (Fig 5I).
Taken together, we concluded that pLORF1 is involved in viral replication in bursal B cells and in Md5-induced bursal atrophy, but not in tumor formation.
Characterization of the LORF1 gene product
To characterize the protein features of pLORF1, the functional domains were analyzed using InterPro (https://www.ebi.ac.uk/interpro/), motifs were identified with MotiFinder (https://www.genome.jp/tools/motif/), and lipid modifications were examined using GPS-Palm [53] (http://gpspalm.biocuckoo.cn/index.php). pLORF1 is predicted to contain two transmembrane domains, two motifs as observed in HSV-1 tegument proteins (VP11/12 and pUL36), and three palmitoylation sites, presented as a structural diagram (Fig 6A), indicating that pLORF1 may share similar features to a tegument protein. Studies show that tegument proteins of HSV-1, including VP22, VP11/12, pUL11, and pUL51, associate with cellular membranes [54–60]. The palmitoylation of pUL11 is essential for its targeting to the Golgi apparatus and membrane binding [60]. To examine the interaction of pLORF1 with membranes, lysates from DEF cells transfected with the Flag-tagged pLORF1 plasmid were analyzed using a membrane flotation assay. The lysates were mixed with sucrose to produce a 72% sucrose mixture, which was then loaded sequentially with 65% and 10% sucrose layers. Due to the buoyant density of membranes, ultracentrifugation resulted in the flotation of membrane-associated proteins to the interface between the 65% and 10% sucrose layers (Fig 6B). Western blot analysis of the membrane flotation gradients revealed that pLORF1 was present in fractions 1 to 5, along with a significant amount of VP22 and a small quantity of GFP protein detected (Fig 6C). The results suggest that pLORF1 expressed within transfected cells is associated with the cellular membranes.
(A) Schematic diagram of pLORF1. (B and C) Membrane flotation analysis was conducted as described in the “Materials and Methods.” (B) Image of a 72%-65%-10% sucrose gradient of extracts from Flag-pLORF1-transfected DEF cells after ultracentrifugation. The mass of floated cellular membranes is marked by a black arrow. (C) Gradient fractions were analyzed using Western blotting with anti-Flag or anti-GFP antibodies. Fractions 1–5 correspond to the 65%-10% interface, which contains cellular membranes, while fractions 10–12 contain non-membrane-associated proteins. (D) Cellular localization of GFP-tagged pLORF1, VP22, VP11/12, pUL11, and pUL51. 293T cells were transfected with the indicated plasmids for 24 hours, then fixed and mounted in DAPI (blue). (E) Cellular localization of Flag-tagged pLORF1, VP22, VP11/12, pUL11, and pUL51. DF-1 cells were transfected with the indicated plasmids for 24 hours, followed by fixation and staining with anti-Flag antibody and Alexa 488-conjugated goat anti-mouse IgG antibody. (F) Cellular localization of pLORF1 in cells infected with rMDV. CEFs were infected with rMd5-GFP-pLORF1 for 3 days, fixed, mounted in DAPI, and analyzed by confocal microscopy. (G) Cellular localization of HA-tagged pLORF1 in cells infected with rMDV. CEFs were infected with rMd5-HA-pLORF1 for 3 days, fixed, and stained with anti-HA antibody and Alexa 488-conjugated goat anti-rabbit IgG antibody (green), followed by staining with anti-pLORF1 antibody and Alexa 555-conjugated goat anti-mouse IgG antibody (red). Scale bar: 10 μm (D and G); 20 μm (E and F).
Previous studies have shown that the tegument proteins VP22, VP11/12, pUL11, and pUL51 display a distinct punctate pattern in the cytoplasm of cells infected with HSV-1 or transfected with these proteins. This pattern resembles the trans-Golgi network (TGN) or TGN-derived vesicles, which are involved in tegument formation and envelopment [55–60]. To determine the subcellular localization of pLORF1, 293T cells were transfected with a plasmid expressing GFP-pLORF1. Confocal imaging revealed that GFP-pLORF1 displayed punctate perinuclear localization within the cytoplasm, similar to GFP-tagged VP22, VP11/12, pUL11, or pUL51 (Fig 6D). Similarly, it also showed punctate localization in the cytoplasm of DF-1 cells transfected with Flag-pLORF1 (Fig 6E). To further examine the cellular localization of pLORF1, recombinant viruses expressing GFP-tagged pLORF1 (rMd5-GFP-pLORF1) or HA-tagged pLORF1 (rMd5-HA-pLORF1) were generated (S5A Fig). The growth curves showed that both viruses proliferated at a rate comparable to that of the parental virus (S5B Fig). Moreover, CEFs infected with rMd5-GFP-pLORF1 displayed green fluorescent plaques, and confocal imaging revealed punctate localization of pLORF1 in the cytoplasm (Fig 6F). Furthermore, a similar localization pattern of HA-pLORF1 was also observed in rMd5-HA-pLORF1-infected CEFs by immunofluorescence analysis, co-immunostained with anti-HA and anti-pLORF1 antibodies (Fig 6G).
It is well known that the tegument of herpesvirus is an organized structure formed by a dense network of interactions among tegument proteins, capsid proteins, and membrane proteins [61,62]. To investigate the interaction between pLORF1 and MDV structural proteins, co-immunoprecipitation assays were performed by co-transfecting Flag-tagged pLORF1 with HA-tagged VP22, VP11/12, VP13/14, or VP19C in DF-1 cells. The results showed the presence of tegument proteins (VP11/12, VP22, VP13/14) and capsid protein (VP19C) in the anti-Flag antibody-immunoprecipitated pLORF1 protein complex (Fig 7A). In addition, we confirmed the ability of pLORF1 to bind with homologous proteins by constructing plasmids expressing HA-tagged counterparts in the alphaherpesvirus HSV-1 and using the gammaherpesvirus EBV tegument protein BGLF2 as a negative control. The immunoprecipitation results showed that pLORF1 interacted with HSV-1 VP22, VP11/12, VP13/14, and VP19C, but not with EBV BGLF2 (Fig 7B). Furthermore, recombinant MDV viruses carrying HA-tagged VP22, VP11/12, VP13/14, and VP19C were successfully generated, respectively (S5C Fig). All recombinant viruses were rescued and exhibited growth rates comparable to that of the parental virus (S5D Fig). To further confirm the interaction between pLORF1 and these proteins during viral infection, CEFs were transfected with Flag-pLORF1, followed by infection with rMd5 expressing HA-tagged proteins, and then immunoprecipitated using an anti-Flag antibody. Results showed that HA-tagged VP22, VP11/12, VP13/14, and VP19C were detected in the anti-Flag (pLORF1) immunocomplex (Fig 7C). Finally, we examined the subcellular localization of pLORF1 and MDV proteins during infection. CEFs infected with rMd5-HA-pLORF1 or rMd5-HA-VP22 viruses were immunostained with anti-HA (pLORF1, green) and anti-VP22 (red) antibodies, or anti-pLORF1 (green) and anti-HA (VP22, red) antibodies. Confocal imaging showed that the endogenously expressed pLORF1 colocalized with VP22 and exhibited a punctate localization in the cytoplasm (Fig 7D and S6 Fig, upper two panels). Similar results were observed in CEFs infected with rMd5-HA-VP11/12, rMd5-HA-VP13/14, and rMd5-HA-VP19C, where pLORF1 exhibited punctate colocalization with VP11/12, VP13/14, and VP19C in the cytoplasm (Fig 7D and S6 Fig, bottom three panels). Taken together, these results suggest that pLORF1 shares similar features to certain tegument proteins.
(A-C) DF-1 cells (A) were co-transfected with Flag-tagged pLORF1 and HA-tagged MDV-1 proteins (VP22, VP11/12, VP13/14, or VP19C) for 36 hours. 293T cells (B) were co-transfected with Flag-tagged pLORF1 and HA-tagged HSV-1 proteins (VP22, VP11/12, VP13/14, or VP19C) or EBV BGLF2 for 36 hours. CEFs (C) were transfected with Flag-tagged pLORF1 for 6 hours and subsequently infected with rMd5-HA-VP22, rMd5-HA-VP11/12, rMd5-HA-VP13/14, or rMd5-HA-VP19C for 2 days. Co-immunoprecipitation assays were performed using whole cell lysates with anti-Flag antibody or control IgG. Immunocomplexes were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. (D) Colocalization of pLORF1 with rMDV structural proteins. CEFs were infected with rMd5-HA-pLORF1, rMd5-HA-VP22, rMd5-HA-VP11/12, rMd5-HA-VP13/14, or rMd5-HA-VP19C for 3 days, then fixed and stained with anti-HA antibody and either Alexa 488 or Alexa 555-conjugated goat anti-rabbit IgG, followed by staining with anti-pLORF1 antibody and either Alexa 555 or Alexa 488-conjugated goat anti-mouse IgG.
Discussion
Marek’s disease (MD), caused by Marek’s disease virus (MDV), poses significant economic challenges to the poultry industry worldwide due to its highly contagious and oncogenic nature [5]. Despite the availability of vaccines, MDV continues to evolve, necessitating the development of improved next-generation vaccines. However, our understanding of the mechanisms underlying MDV-induced tumorigenesis and immunosuppression remains incomplete. With MDV encoding more than 100 genes, most of which have unknown functions, exploring the roles of these genes is crucial. Among these genes, MDV LORF1 is unique to MDV-1, lacking homologs in other herpesviruses [30]. However, its function in MDV pathogenesis remains largely unknown [47]. In this study, we investigated the function of LORF1 in MDV infection both in vitro and in vivo using genetically modified Md5-deficient in pLORF1 expression. Our findings suggest that pLORF1 is involved in viral replication and MDV-induced bursal atrophy, but not in tumor development.
The complete genome of the MDV-1 Md5 strain was cloned as an infectious BAC using a visible selection marker (RFP) and the BAC vector pBeloBAC11 (Fig 1). Previously reported MDV-BAC constructions exclusively relied on the expression of the Eco-gpt gene and selection media containing mycophenolic acid, xanthine, and hypoxanthine to enrich recombinant viruses [9–18]. In this study, we introduced the RFP expression cassette for the first time in MDV-BAC construction, which simplifies the purification of positive clones. Additionally, the transfer vector also includes two 34-bp loxP sites flanking the BAC and RFP fragment, enabling the rapid excision of BAC vector sequences using the Cre/loxP homologous recombination system (Fig 1 and S1 Fig). Transfection of the DNA prepared from BAC clones and the Cre expression vector in CEFs resulted in the rescue of infectious viruses with plaque morphology and growth kinetics indistinguishable from those of the parental Md5 virus (Fig 2B and 2C). In animal studies, BAC-derived rMd5 viruses exhibited high pathogenicity and oncogenicity, as well as horizontal transmissibility, comparable to the parental Md5 virus in vivo (Fig 2 and S1 Fig). During the experiments, we observed that the majority of chickens infected with rMd5 developed gross tumors and/or bursal atrophy, with a few exceptions (S1D Fig). Indeed, a previous report showed that 14.3% of maternal antibody-positive chickens and 2.6% of negative chickens showed no bursal atrophy when infected with the vv+MDV 648A strain [52]. In addition, H&E staining revealed lymphocyte infiltration of rMd5-infected liver tissues from chickens without obvious tumor nodules collected at 28 dpi (S1F Fig), consistent with previous reports that 20% of Md5-infected chickens developed obvious tumors [27,63].
Using the Red-mediated recombination system, we constructed the pLORF1-deficient virus, rMd5ΔLORF1, by creating premature stop codons right after the translation start site due to a 2-bp frameshift deletion within the LORF1 gene, along with the revertant virus, rMd5-reLORF1 (Fig 3A). MD pathogenesis involves three main stages: an early cytolytic phase (2–7 dpi) mainly targeting and infecting B lymphocytes [64,65], activating CD4+ T cells; a latent phase with no viral antigen expression; and a second cytolytic phase leading to rapid proliferation of latently infected and transformed lymphocytes, resulting in tumor formation in tissues [5,66,67]. In animal studies, we observed that the loss of pLORF1 expression attenuated rMd5-induced pathogenesis, delaying weight loss and bursal atrophy compared to the parental and revertant viruses (Fig 4A–4C and S3 Fig). Interestingly, the viral load of rMd5ΔLORF1 was notably lower than that of rMd5 or rMd5-reLORF1 in the bursa, but not in the spleen by 35 dpi (Fig 4D). The loss of pLORF1 did not affect rMd5 replication in vitro (Fig 3E). There was almost no replication of rMd5ΔLORF1 in the bursa and no damage to bursal structure at the early stage of infection (7 dpi) (Fig 5A–5D), but only moderate replication of rMd5ΔLORF1 concomitantly with lymphocytic infiltration at the late stage of infection (42 dpi) (Fig 5E–5G). The bursa of Fabricius is a primary lymphoid organ in birds responsible for amplifying and differentiating B lymphoid progenitors within its follicular microenvironment. Our study showed that the loss of pLORF1 abolished the capability of rMd5 to replicate in bursal B cells at 7 dpi (Fig 5I).
Efficient virus replication in the host is a prerequisite for disease and tumor formation. However, the low level of viral replication in the bursa due to pLORF1 deficiency did not affect the ability of rMd5 to induce lymphomas (Fig 5E and 5F, and S4 Fig). As previously reported, in the absence of B cells, MDV can readily replicate in macrophages [68,69], T cells [65,70,71], and natural killer cells [72], compensating for the loss of B cells during lytic replication. MD pathogenesis and tumor formation were not altered in infected chickens that lack mature and peripheral B cells [73]. These results suggest that pLORF1 is essential for early cytolytic infection in bursal B cells, but not for tumor formation. Integration of the MDV genome into latently infected and transformed cells has been shown to be essential for effective tumor formation [74,75]. Therefore, indistinguishable viral loads (genome copies) of recombinant viruses in the bursa at 42 dpi may be caused by infiltration with lymphocytes bearing viral genomes.
To date, there is only limited information available regarding the MDV-1 unique gene LORF1. Based on prediction data, pLORF1 contains two transmembrane domains, two tegument motifs (VP11/12 and pUL36), and at least three palmitoylation sites (Fig 6A). Importantly, we found that pLORF1 can bind to the cellular membrane, has a punctate distribution in the cytoplasm, and interacts with tegument proteins (VP22, VP11/12, VP13/14) and the capsid protein (VP19C) (Figs 6 and 7). The ability of tegument proteins to target membranes helps transport viral components and recruit essential host cell factors to the specific assembly site for forming new infectious particles [76–78]. Previous studies have shown that the tegument proteins VP22, VP11/12, pUL11, and pUL51 display a distinct punctate pattern in the cytoplasm of cells. This punctate localization resembles the trans-Golgi network (TGN) and TGN-derived vesicles [55–57,78], where tegument proteins direct HSV-1 virion assembly by acquiring the envelope and nucleocapsid [79–81], which have been extensively studied with HSV-1 proteins VP22, VP11/12, pUL11, and pUL51. Given that palmitoylation enhances the hydrophobicity of proteins, resulting in alterations to protein conformation, stability, membrane association, localization, and binding properties [82], it is possible that pLORF1 binds to TGN membranes through its transmembrane domains and palmitoylation, thereby recruiting nucleocapsids and tegument proteins for viral packaging. Here, we would also like to note that the conserved amino acid residues shared between pLORF1 and the tegument proteins VP11/12 or pUL36, which have not yet been characterized in terms of any biological function, warrant further investigation to elucidate the specific roles of these motifs. In addition, the role of pLORF1 during the specific stage of virus replication needs to be further explored as well.
In this study, we found that the MDV-1 unique gene LORF1 is a nonessential gene for replication, but it plays an important role in bursal atrophy, indicating its potential for the development of a gene deletion vaccine for MD. The Meq deletion virus (rMd5ΔMeq) conferred superior protection against challenge with vv+ MDV strains, making it a promising vaccine candidate [28,29]. However, rMd5ΔMeq retains the ability to induce lymphoid organ atrophy and lower body weights (Fig 4C and S4B Fig) in maternal antibody-negative chickens [83], rendering it unsuitable for approval as a vaccine. Attempts to eliminate rMd5ΔMeq-induced lymphoid organ atrophy through serial cell culture passages led to an attenuated virus with decreased protective efficacy [84]. An alternative strategy involved introducing known secondary mutations in the MDV genome by deleting or mutating a second gene related to virus replication. Studies have demonstrated that introducing a point mutation in UL5 (a helicase-primase subunit), codon deoptimization of UL54 (a multifunctional ICP27-like protein), ablation of UL23 (thymidine Kinase), deletion of pp38 (a phosphorylated protein), LORF9 (a unique MDV gene), or vIL-8 (an interleukin-8 homolog) led to reduced lymphoid organ atrophy and provided partial or comparable protection to commercial vaccines against vv or vv+ MDV strains [63,85–89]. These double deletion viruses exhibited comparable replication capacity to the parental virus in vitro but were significantly less replicative and pathogenic in vivo, while inducing varying degrees of immune response. This suggests that the protective efficacy of MD vaccines is deeply associated with their replication in vivo. Our findings suggest that pLORF1 deficiency inhibits MDV replication in the bursa, preventing early-stage bursal atrophy and body weight loss. Defective pLORF1 expression may prevent immunosuppressive effects while inducing maximum disease protection by the rMd5ΔMeq virus. Further studies will validate the efficacy of the Meq&LORF1-deleted vaccine.
Materials and methods
Ethics statement
All animals and experimental protocols were approved by the Animal Welfare and Ethics Committee of Nanjing Agricultural University (SYXK(Su)2021-0086).
Cells, viruses, and reagents
293T and DF-1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) supplemented with 8% fetal bovine serum (FBS; PAN Biotech, Germany) and 1% penicillin and streptomycin at 37°C with 5% CO2. Chicken embryo fibroblasts (CEFs) were prepared from 10-day-old specific-pathogen-free (SPF) chicken embryos and maintained in DMEM containing 5% FBS (Gibco, USA). Duck embryo fibroblasts (DEFs) were prepared from 10-day-old duck embryos and cultured in DMEM containing 10% FBS (Gibco, USA). The virulent MDV Md5 strain (GenBank: AF243438.1; kindly provided by Dr. Xunhai Zhang, Anhui Science and Technology University, China) was propagated in CEFs.
Antibodies
A mouse anti-pLORF1 polyclonal antibody was generated by using the pET30a vector to express the C-terminal portion of pLORF1 (aa 174 to 333) as a fusion protein with (His)6 tags at both ends, which was purified using Ni-NTA agarose (Sangon Biotech, China) and injected into BALB/c mice to generate antisera. A rabbit anti-HA antibody was purchased from Cell Signaling Technology (USA). Mouse anti-Flag, horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG antibodies were purchased from Sigma-Aldrich (USA). Mouse anti-Bu-1, Alexa Fluor-conjugated goat anti-mouse and anti-rabbit IgG antibodies were purchased from Thermo Fisher Scientific (USA). A mouse anti-VP22 monoclonal antibody (clone 3F7) was provided by Dr. Hongjun Chen from the Shanghai Veterinary Research Institute in China.
Construction of transfer vectors
A 2.1-kbp NotI-BamHI fragment and a 3.0-kbp HindIII-KpnI fragment flanking the MDV-1 US2 gene were amplified by PCR using the primers listed in Table 1. Both fragments were subsequently cloned into pBluescript II KS(+) to yield pBlue-US2A-US2B. A 2.5-kbp SalI fragment of pBS302 (Addgene, USA) was amplified by PCR using primers provided in Table 1 and then circularized as pBS302-CB. To construct pBS302-BAC-RFP, a 6.4-kbp SalI fragment of pBeloBAC11 (NEB, USA) and a 2.1-kbp NotI-BamHI fragment of the NEDD8 HDR plasmid (Santa Cruz, USA) containing the red fluorescent protein (RFP) gene and the puromycin resistance gene (both sites were blunt-ended) were cloned into the SalI and HpaI sites of pBS302-CB, respectively. To generate pBlue-US2-BAC-RFP, a NotI fragment of pBS302-BAC-RFP containing the BAC vector and RFP & puromycin cassette (the NotI sites were blunt-ended) was cloned into the SmaI site in pBlue-US2A-US2B.
Construction of Md5 BAC
CEFs were transfected with linearized pBlue-US2-BAC-RFP using jetPRIME transfection reagent (Polyplus, France). At 8 hours post-transfection, the CEFs were inoculated with MDV Md5 at 0.01 PFU/cell. At 3 days post-inoculation, monolayers were harvested and serially diluted on fresh CEFs in 96-well plates. At 7 days post-inoculation, a single red fluorescent plaque was picked from the well. After six rounds of plaque purification, the recombinant virus Md5-BAC-RFP DNA was isolated from infected CEFs using the Hirt method as previously described [90], and then electroporated into E. coli DH10B cells. Chloramphenicol-resistant colonies were analyzed for the integrity of Md5 genomes by restriction fragment length polymorphism (RFLP) analysis. The BAC DNA was digested with EcoRI, BamHI, HindIII, or SacI and separated on 0.8% agarose gels. The profiles obtained were compared to the simulated profiles generated by SnapGene software. For the infectivity of pMd5, BAC DNA was transfected into CEFs, and the growth of the rescued virus rMd5-BAC-RFP was observed with the aid of a fluorescence microscope.
Removal of BAC sequence
The loxP-flanked BAC and RFP & puromycin sequences within the infectious clones were removed by co-transfection of pMd5 DNA and a Cre recombinase expression vector (pcDNA3-Cre: GFP). Removal of the exogenous sequences was confirmed by analyzing recombinant virus rMd5 stocks using PCR amplification. Genomic regions of viral, BAC, RFP, and junctional DNA were amplified using the primers provided in Table 1.
Construction of recombinant viruses
To generate the recombinant virus rMd5ΔLORF1, in which LORF1 harbored a frameshift due to a 2-bp deletion, the two-step Red-mediated mutagenesis procedure was conducted using E. coli GS1783 (kindly provided by Dr. Nikolaus Osterrieder, Freie Universität Berlin, Germany) containing pMd5, following the previously described protocol [51], with the primers listed in Table 1. The revertant virus rMd5-reLORF1, the full-length Meq-deleted virus rMd5ΔMeq, and several tagged viruses (rMd5-GFP-pLORF1, rMd5-HA-pLORF1, rMd5-HA-VP11/12, rMd5-HA-VP22, rMd5-HA-VP13/14, and rMd5-HA-VP19C) were generated using the same strategy, except for the primers listed in Table 1.
Growth curve analysis
The growth curves of recombinant viruses were determined as previously described [9]. Briefly, CEFs pre-seeded in 6-well plates were infected with 100 PFU of each virus. At 0, 1, 2, 3, 4, and 5 dpi, the infected cells were trypsinized and serially diluted on fresh CEFs pre-seeded in 6-well plates, and the plaques were counted at 7 dpi.
Chicken experiment
One-day-old SPF chickens were randomly divided into groups (n = 25) and intraperitoneally injected with 1,000 PFU of either Md5 or rMd5/14-20, and contact chickens (n = 3) were kept in the same isolator as the Md5 or rMd5 group. A negative control group (n = 10) was injected intraperitoneally with DMEM. The birds were monitored daily, and mortality rates were recorded. To assess viral pathogenesis and histopathology (gross and histological lesions), one bird from the control group and two birds from the Md5 or rMd5 group were randomly selected and euthanized at 28 dpi or 42 dpi. The spleens and bursas were dissected and photographed. The liver and heart tissues were dissected, fixed in neutral buffered 4% paraformaldehyde, processed routinely for paraffin embedding, sectioned, and stained with hematoxylin-eosin (H&E). The experiment was terminated at 54 dpi. To detect the transmission of rMd5, three contact birds from the Md5 or rMd5 group were euthanized, the spleen tissues were collected for DNA preparation, and the liver tissues were dissected for H&E staining. Meanwhile, moribund and surviving birds were humanely euthanized and assessed for gross and histological lesions.
To characterize recombinant rMd5ΔLORF1, a total of 149 one-day-old SPF chickens were randomly divided into five groups: rMd5ΔLORF1 group (35 chickens, 20 for survival rate, 15 for sampling), rMd5-reLORF1 group (35 chickens, 20 for survival rate, 15 for sampling), rMd5ΔMeq group (35 chickens, 20 for survival rate, 15 for sampling), rMd5 group (28 chickens, 20 for survival rate, 8 for sampling), and a negative control group (16 chickens, 8 for survival rate, 8 for sampling). The first four groups were intraperitoneally injected with 1,000 PFU of each virus, while the control group was injected with DMEM. Chickens were monitored daily, and mortality rates were recorded from 5 to 60 dpi. At 7, 14, 21, 28, 35, and 42 dpi, one bird from the control or rMd5 group and two birds from the rMd5ΔLORF1, rMd5-reLORF1, or rMd5ΔMeq groups were humanely euthanized. The spleens and bursas were dissected for DNA preparation, and the latter were also photographed and weighed. Liver and bursal tissues were fixed for histopathological analysis.
Immunohistochemistry (IHC)
Bursas were harvested at 7 and 42 dpi, and fixed in 4% paraformaldehyde. Tissue sections underwent microwave antigen repair and blocking, followed by overnight incubation at 4°C with either anti-VP22 antibody or Bu-1 antibodies. The sections were then incubated for 50 minutes at room temperature with HRP-conjugated goat anti-mouse IgG antibody or Alexa-conjugated goat anti-mouse IgG antibodies. HRP was visualized using the DAB Kit (Wuhan Servicebio Technology, China), and nuclei were stained with either hematoxylin or DAPI. Images were captured using a microscope scanner (Grundium Ocus 40, Finland) or a confocal microscope (Nikon Eclipse Ti, A1, Japan).
Plasmids
To generate GFP-tagged pLORF1, VP22 (encoded by the UL49 gene), VP11/12 (encoded by the UL46 gene), pUL11, or pUL51, HA-tagged VP22, VP11/12, VP13/14 (encoded by the UL47 gene), or VP19C (encoded by the UL38 gene), and 2xFlag-tagged pLORF1, DNA fragments were amplified from the Md5 genome and cloned into pEGFP-C1, pcDNA3-HA, and pcDNA3-2xFlag using their respective restriction sites. HSV-1 VP22, VP11/12, VP13/14, and VP19C were amplified from the F strain genome (GenBank: KM222724.1) and cloned into the pCAGGS-N-HA vector. EBV BGLF2 (encoded by the UL16 gene; GenBank: NC_007605.1) was synthesized and cloned into the pCAGGS-N-HA vector by GenScript (China). The primers used in this study are listed in S2 Table.
Membrane flotation gradient centrifugation
The protocol for the analysis of membrane-associated proteins by sucrose density gradient flotation was performed as previously described [56,57]. Briefly, DEF cells were transfected with pcDNA3-2xFlag-pLORF1, pcDNA3-2xFlag-VP22, or pEGFP-C1 for 24 hours. The cells were collected and resuspended in a hypotonic lysis buffer before being lysed by passing them through a 25-gauge needle. Post-nuclear supernatants (PNS) were obtained through low-speed centrifugation. The PNS obtained was loaded onto the bottom of a non-continuous 72%-65%-10% sucrose gradient and centrifuged at 150,000 × g at 4°C for 18 hours. The gradients were fractionated from the bottom and then mixed with trichloroacetic acid (TCA) for precipitation. After centrifugation, the pellets were washed once with ethanol, solubilized with 2×SDS sample buffer, and analyzed by Western blot.
Confocal imaging
293T or DF-1 cells were pre-seeded on coverslips and transfected with GFP-tagged or Flag-tagged plasmids for 24 hours. CEFs were infected with MDV for 3 days. The coverslip cells were fixed and permeabilized with 4% formaldehyde and 0.1% Triton X-100 at 37°C for 30 minutes, washed with glycine-PBS, and blocked with 3% BSA in PBS at 37°C for 30 minutes. They were incubated with a primary antibody (1:200) at 37°C for 1 hour, followed by a secondary antibody (1:500) for 30 minutes. Nuclei were visualized with DAPI in an anti-fade DABCO solution and examined using a confocal microscope.
Co-immunoprecipitation and Western blotting
293T or DF-1 cells were co-transfected with pcDNA3-2xFlag-LORF1 and HA-tagged plasmids for 36 hours. CEFs were first transfected with pcDNA3-2xFlag-LORF1 and infected with HA-tagged rMDV for 48 hours. Cells were collected in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, and 0.5% NP-40 supplemented with protease inhibitor cocktails) and incubated with an anti-Flag antibody, along with protein A/G magnetic beads, for 4 hours at 4°C. Mouse IgG was used as a negative control. The beads were washed five times with ice-cold lysis buffer. The precipitation was mixed with 2×SDS sample buffer and boiled for 10 minutes at 98°C. After centrifugation, the supernatant was used for Western blot analysis.
Statistical analysis
All experiments were performed at least three times unless otherwise noted. The data are presented as the means ± standard deviations (SD). Statistical significance between groups was determined by a Student’s t-test using GraphPad Prism 7.0 software. A p-value of < 0.05 was considered statistically significant.
Supporting information
S1 Table. Bursa/body weight ratios in experimental chickens at different time points post-inoculation.
https://doi.org/10.1371/journal.ppat.1012891.s001
(DOCX)
S2 Table. Primers used for the construction of expression plasmids.
https://doi.org/10.1371/journal.ppat.1012891.s002
(DOCX)
S1 Fig. Analysis of the infectious clone pMd5 BACs and BAC-derived viruses.
(A) The image shows a simulated experiment using the SnapGene software. Md5 or pMd5 BAC genomes are digested with EcoRI, BamHI, HindIII, or SacI on a 0.8% agarose gel. The bands marked with asterisks indicate the insertion of the transfer vector. (B) Recombinant viruses rMd5-BAC-RFP clones 14–4 and 14–20 were generated by transfecting CEFs with pMd5 DNA. At 5 days post-transfection, plaques expressing RFP were observed under fluorescence microscopy. (C) Recombinant viruses rMd5/14-4 and rMd5/14-20 were obtained by co-transfecting CEFs with pMd5 and Cre expression plasmid. The junction fragments (US2Aj and US2Bj), inserted genes (RFP and sopA), and viral genes (VP22) in mock-, Md5-, rMd5-BAC-RFP, and rMd5-infected CEFs at 3 dpi were amplified by PCR. (D-G) The animal experiment was performed as described in “Materials and Methods.” (D) Images of the bursas and spleens from chickens of the Md5 or rMd5 group at 28 dpi. (E) Images of the livers from chickens in each group at 42 dpi. White arrows indicate tumor nodules in the livers. (F) Liver tissues infected with rMd5 at 28 dpi were stained with H&E. (G) Liver tissues from contact chickens at 54 dpi in each group were stained with H&E. Scale bar: 20 μm (F and G).
https://doi.org/10.1371/journal.ppat.1012891.s003
(TIF)
S2 Fig. Construction of the Meq-deleted virus.
(A) The linear MDV-1 Md5 genome and a portion of the repeat regions (TRL and IRL) encoding genes LORF11 to vIL8 are depicted. (B) Schematic diagrams of the rMd5ΔMeq virus with a full-length deletion of the Meq gene. (C) DNA analysis of pMd5 and pMd5ΔMeq genomes. DNAs purified from BACs containing E. coli were digested with HindIII and separated on a 0.8% agarose gel. The bands that appeared after the deletion of the Meq gene are marked with arrows. (D) PCR amplification result of the Meq locus in pMd5 or pMd5ΔMeq. (E) CEFs infected with 100 PFU of rMd5 or rMd5ΔMeq were harvested at the indicated time points and subsequently seeded onto fresh CEFs. Viral plaques were counted at 7 dpi. Each value represents the mean of two independent duplicates.
https://doi.org/10.1371/journal.ppat.1012891.s004
(TIF)
S3 Fig. Loss of pLORF1 attenuates MDV-induced bursal atrophy.
One-day-old SPF chickens were intraperitoneally inoculated with 1,000 PFU of rMd5, rMd5ΔLORF1, or rMd5-reLORF1, and DMEM as a negative control. The chickens were kept in isolators for daily monitoring of MD symptoms or death. (A) Images of bursas of four representative dead chickens from each group at the indicated time points. (B) The histogram of the body weights of chickens in each group at 7, 14, 21, 28, 35, and 42 dpi. Results represent the mean value, with error bars indicating the standard error of the mean. (C) Images of chicken bursas from each group at the indicated time points, as in B.
https://doi.org/10.1371/journal.ppat.1012891.s005
(TIF)
S4 Fig. Histopathological examination of chicken liver tissues infected with recombinant viruses.
The liver tissues from chickens were mock-treated or inoculated with 1,000 PFU of rMd5, rMd5ΔLORF1, or rMd5-reLORF1 and euthanized at 35 dpi. (A) Focal infiltration of lymphoid tumor cells in the liver tissues is highlighted by white arrows (H&E). (B) A higher magnification of areas indicated by red boxes in A. Scale bar: 1 mm (A), 20 μm (B).
https://doi.org/10.1371/journal.ppat.1012891.s006
(TIF)
S5 Fig. Construction of recombinant viruses expressing GFP-tagged or HA-tagged viral proteins.
(A and C) RFLP analysis of HindIII-digested BAC DNAs, including pMd5, pMd5-HA-pLORF1, pMd5-GFP-pLORF1, pMd5-HA-VP22, pMd5-HA-VP11/12, pMd5-HA-VP13/14, and pMd5-HA-VP19C. (B and D) The growth curve of recombinant viruses, including rMd5, rMd5-HA-pLORF1, rMd5-GFP-pLORF1, rMd5-HA-VP22, rMd5-HA-VP11/12, rMd5-HA-VP13/14, and rMd5-HA-VP19C. Each value represents the mean of two independent duplicates.
https://doi.org/10.1371/journal.ppat.1012891.s007
(TIF)
S6 Fig. Colocalization of pLORF1 with MDV structural proteins.
CEFs were infected with rMd5-HA-pLORF1, rMd5-HA-VP22, rMd5-HA-VP11/12, rMd5-HA-VP13/14, or rMd5-HA-VP19C for 3 days. The cells were fixed and stained with anti-HA antibody, followed by either Alexa 488 or Alexa 555-conjugated goat anti-rabbit IgG. Subsequently, the cells were treated with anti-pLORF1 antibody and either Alexa 555 or Alexa 488-conjugated goat anti-mouse IgG. Zoomed-in views of representative cells, indicated by white boxes, are shown in Fig 7D.
https://doi.org/10.1371/journal.ppat.1012891.s008
(TIF)
S1 Data. Excel containing the numerical data underlying Fig 2.
https://doi.org/10.1371/journal.ppat.1012891.s009
(XLSX)
S2 Data. Excel containing the numerical data underlying Fig 3.
https://doi.org/10.1371/journal.ppat.1012891.s010
(XLSX)
S3 Data. Excel containing the numerical data underlying Fig 4.
https://doi.org/10.1371/journal.ppat.1012891.s011
(XLSX)
S4 Data. Excel containing the numerical data underlying S2 Fig.
https://doi.org/10.1371/journal.ppat.1012891.s012
(XLSX)
S5 Data. Excel containing the numerical data underlying S5 Fig.
https://doi.org/10.1371/journal.ppat.1012891.s013
(XLSX)
Acknowledgments
We would like to acknowledge Dr. Yingjun Lv (NJAU Nanjing) for his generous assistance in conducting and analyzing the pathological examination of chicken tissues. We also greatly thank Dr. Jakob Trimpert (FU Berlin), Dr. Dusan Kunec (FU Berlin), and Dr. Guanqun Liu (VIDO-InterVac Saskatoon) for their technical assistance.
References
- 1. Nair V. Spotlight on avian pathology: Marek’s disease. Avian Pathol. 2018;47(5):440–2. pmid:29882420
- 2. Kingham BF, Zelnık V, Kopáček J, Majerčiak V, Ney E, Schmidt CJ. The genome of herpesvirus of turkeys: comparative analysis with Marek’s disease viruses. The Journal of general virology. 2001;82(Pt 5):1123–35. pmid:11297687
- 3. Davison AJ. Evolution of the herpesviruses. Veterinary microbiology. 2002;86(1–2):69–88. pmid:11888691
- 4. Davison A. Comments on the phylogenetics and evolution of herpesviruses and other large DNA viruses. Virus research. 2002;82(1–2):127–32. pmid:11885939
- 5. Osterrieder N, Kamil JP, Schumacher D, Tischer BK, Trapp S. Marek’s disease virus: from miasma to model. Nat Rev Microbiol. 2006;4(4):283–94. pmid:16541136
- 6. Epstein MA. Historical background. Philos Trans R Soc Lond B Biol Sci. 2001;356(1408):413–20. pmid:11313002
- 7. Biggs PM, Nair V. The long view: 40 years of Marek’s disease research and Avian Pathology. Avian Pathol. 2012;41(1):3–9. pmid:22845316
- 8. Witter RL. Increased virulence of Marek’s disease virus field isolates. Avian Dis. 1997;41(1):149–63. pmid:9087332
- 9. Schumacher D, Tischer BK, Fuchs W, Osterrieder N. Reconstitution of Marek’s disease virus serotype 1 (MDV-1) from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant. J Virol. 2000;74(23):11088–98. pmid:11070004
- 10. Petherbridge L, Howes K, Baigent SJ, Sacco MA, Evans S, Osterrieder N, et al. Replication-competent bacterial artificial chromosomes of Marek’s disease virus: novel tools for generation of molecularly defined herpesvirus vaccines. J Virol. 2003;77(16):8712–8. pmid:12885890
- 11. Petherbridge L, Brown AC, Baigent SJ, Howes K, Sacco MA, Osterrieder N, et al. Oncogenicity of virulent Marek’s disease virus cloned as bacterial artificial chromosomes. J Virol. 2004;78(23):13376–80. pmid:15542691
- 12. Niikura M, Dodgson J, Cheng H. Direct evidence of host genome acquisition by the alphaherpesvirus Marek’s disease virus. Archives of virology. 2006;151(3):537–49. pmid:16155725
- 13. Baigent SJ, Petherbridge LJ, Smith LP, Zhao Y, Chesters PM, Nair VK. Herpesvirus of turkey reconstituted from bacterial artificial chromosome clones induces protection against Marek’s disease. The Journal of general virology. 2006;87(Pt 4):769–76. pmid:16528024
- 14. Petherbridge L, Xu H, Zhao Y, Smith LP, Simpson J, Baigent S, et al. Cloning of Gallid herpesvirus 3 (Marek’s disease virus serotype-2) genome as infectious bacterial artificial chromosomes for analysis of viral gene functions. Journal of virological methods. 2009;158(1–2):11–7. pmid:19187788
- 15. Sun A, Lawrence P, Zhao Y, Li Y, Nair VK, Cui Z. A BAC clone of MDV strain GX0101 with REV-LTR integration retained its pathogenicity. Chinese Science Bulletin. 2009;54(15):2641–7.
- 16. Smith LP, Petherbridge LJ, Baigent SJ, Simpson J, Nair V. Pathogenicity of a very virulent strain of Marek’s disease herpesvirus cloned as infectious bacterial artificial chromosomes. Journal of biomedicine & biotechnology. 2011;2011:412829. pmid:21127705
- 17. Reddy SM, Sun A, Khan OA, Lee LF, Lupiani B. Cloning of a very virulent plus, 686 strain of Marek’s disease virus as a bacterial artificial chromosome. Avian Dis. 2013;57(2 Suppl):469–73. pmid:23901763
- 18. Cui HY, Wang YF, Shi XM, An TQ, Tong GZ, Lan DS, et al. Construction of an infectious Marek’s disease virus bacterial artificial chromosome and characterization of protection induced in chickens. Journal of virological methods. 2009;156(1–2):66–72. pmid:19026690
- 19. Kamil JP, Tischer BK, Trapp S, Nair VK, Osterrieder N, Kung HJ. vLIP, a viral lipase homologue, is a virulence factor of Marek’s disease virus. J Virol. 2005;79(11):6984–96. pmid:15890938
- 20. Brown AC, Baigent SJ, Smith LP, Chattoo JP, Petherbridge LJ, Hawes P, et al. Interaction of Meq protein and C-terminal-binding protein is critical for induction of lymphomas by Marek’s disease virus. Proc Natl Acad Sci U S A. 2006;103(6):1687–92. pmid:16446447
- 21. Trapp S, Parcells MS, Kamil JP, Schumacher D, Tischer BK, Kumar PM, et al. A virus-encoded telomerase RNA promotes malignant T cell lymphomagenesis. The Journal of experimental medicine. 2006;203(5):1307–17. pmid:16651385
- 22. Zhao Y, Xu H, Yao Y, Smith LP, Kgosana L, Green J, et al. Critical role of the virus-encoded microRNA-155 ortholog in the induction of Marek’s disease lymphomas. PLoS pathogens. 2011;7(2):e1001305. pmid:21383974
- 23. Krieter A, Ponnuraj N, Jarosinski KW. Expression of the conserved herpesvirus protein kinase (CHPK) of Marek’s disease alphaherpesvirus in the skin reveals a mechanistic importance for CHPK during interindividual spread in chickens. J Virol. 2020;94(5). pmid:31801854
- 24. Jarosinski KW, Osterrieder N, Nair VK, Schat KA. Attenuation of Marek’s disease virus by deletion of open reading frame RLORF4 but not RLORF5a. J Virol. 2005;79(18):11647–59. pmid:16140742
- 25. Chuard A, Courvoisier-Guyader K, Rémy S, Spatz S, Denesvre C, Pasdeloup D. The tegument protein pUL47 of Marek’s disease virus is necessary for horizontal transmission and is important for expression of glycoprotein gC. J Virol. 2020;95(2). pmid:32999032
- 26. Jarosinski KW, Margulis NG, Kamil JP, Spatz SJ, Nair VK, Osterrieder N. Horizontal transmission of Marek’s disease virus requires US2, the UL13 protein kinase, and gC. J Virol. 2007;81(19):10575–87. pmid:17634222
- 27. Zhu X, Wang L, Gong L, Zhai Y, Wang R, Jin J, et al. LORF9 of Marek’s disease virus is involved in the early cytolytic replication of B lymphocytes and can act as a target for gene deletion vaccine development. J Virol. 2023;97(12):e0157423. pmid:38014947
- 28. Lee LF, Lupiani B, Silva RF, Kung HJ, Reddy SM. Recombinant Marek’s disease virus (MDV) lacking the Meq oncogene confers protection against challenge with a very virulent plus strain of MDV. Vaccine. 2008;26(15):1887–92. pmid:18313812
- 29. Lee LF, Kreager KS, Arango J, Paraguassu A, Beckman B, Zhang H, et al. Comparative evaluation of vaccine efficacy of recombinant Marek’s disease virus vaccine lacking Meq oncogene in commercial chickens. Vaccine. 2010;28(5):1294–9. pmid:19941987
- 30. Tulman ER, Afonso CL, Lu Z, Zsak L, Rock DL, Kutish GF. The genome of a very virulent Marek’s disease virus. J Virol. 2000;74(17):7980–8. pmid:10933706
- 31. Lee LF, Wu P, Sui D, Ren D, Kamil J, Kung HJ, et al. The complete unique long sequence and the overall genomic organization of the GA strain of Marek’s disease virus. Proc Natl Acad Sci U S A. 2000;97(11):6091–6. pmid:10823954
- 32. Tischer BK, Schumacher D, Messerle M, Wagner M, Osterrieder N. The products of the UL10 (gM) and the UL49.5 genes of Marek’s disease virus serotype 1 are essential for virus growth in cultured cells. The Journal of general virology. 2002;83(Pt 5):997–1003. pmid:11961253
- 33. Sun A, Yang S, Luo J, Teng M, Xu Y, Wang R, et al. UL28 and UL33 homologs of Marek’s disease virus terminase complex involved in the regulation of cleavage and packaging of viral DNA are indispensable for replication in cultured cells. Veterinary research. 2021;52(1):20. pmid:33579382
- 34. Anderson AS, Parcells MS, Morgan RW. The glycoprotein D (US6) homolog is not essential for oncogenicity or horizontal transmission of Marek’s disease virus. J Virol. 1998;72(3):2548–53. pmid:9499123
- 35. Schumacher D, Tischer BK, Reddy SM, Osterrieder N. Glycoproteins E and I of Marek’s disease virus serotype 1 are essential for virus growth in cultured cells. J Virol. 2001;75(23):11307–18. pmid:11689611
- 36. Schumacher D, Tischer BK, Trapp S, Osterrieder N. The protein encoded by the US3 orthologue of Marek’s disease virus is required for efficient de-envelopment of perinuclear virions and involved in actin stress fiber breakdown. J Virol. 2005;79(7):3987–97. pmid:15767401
- 37. Liao Y, Lupiani B, Ai-Mahmood M, Reddy SM. Marek’s disease virus US3 protein kinase phosphorylates chicken HDAC 1 and 2 and regulates viral replication and pathogenesis. PLoS pathogens. 2021;17(2):e1009307. pmid:33596269
- 38. Du X, Zhou D, Zhou J, Xue J, Wang G, Cheng Z. Marek’s disease virus serine/threonine kinase Us3 facilitates viral replication by targeting IRF7 to block IFN-β production. Veterinary microbiology. 2022;266:109364. pmid:35144044
- 39. Lupiani B, Lee LF, Cui X, Gimeno I, Anderson A, Morgan RW, et al. Marek’s disease virus-encoded Meq gene is involved in transformation of lymphocytes but is dispensable for replication. Proc Natl Acad Sci U S A. 2004;101(32):11815–20. pmid:15289599
- 40. Parcells MS, Lin SF, Dienglewicz RL, Majerciak V, Robinson DR, Chen HC, et al. Marek’s disease virus (MDV) encodes an interleukin-8 homolog (vIL-8): characterization of the vIL-8 protein and a vIL-8 deletion mutant MDV. J Virol. 2001;75(11):5159–73. pmid:11333897
- 41. Cui X, Lee LF, Reed WM, Kung HJ, Reddy SM. Marek’s disease virus-encoded vIL-8 gene is involved in early cytolytic infection but dispensable for establishment of latency. J Virol. 2004;78(9):4753–60. pmid:15078957
- 42. Cui X, Lee LF, Hunt HD, Reed WM, Lupiani B, Reddy SM. A Marek’s disease virus vIL-8 deletion mutant has attenuated virulence and confers protection against challenge with a very virulent plus strain. Avian Dis. 2005;49(2):199–206. pmid:16094823
- 43. Kaufer BB, Arndt S, Trapp S, Osterrieder N, Jarosinski KW. Herpesvirus telomerase RNA (vTR) with a mutated template sequence abrogates herpesvirus-induced lymphomagenesis. PLoS pathogens. 2011;7(10):e1002333. pmid:22046133
- 44. Engel AT, Selvaraj RK, Kamil JP, Osterrieder N, Kaufer BB. Marek’s disease viral interleukin-8 promotes lymphoma formation through targeted recruitment of B cells and CD4+ CD25+ T cells. J Virol. 2012;86(16):8536–45. pmid:22647701
- 45. Reddy SM, Lupiani B, Gimeno IM, Silva RF, Lee LF, Witter RL. 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. Proc Natl Acad Sci U S A. 2002;99(10):7054–9. pmid:11997455
- 46. Cui ZZ, Lee LF, Liu JL, Kung HJ. Structural analysis and transcriptional mapping of the Marek’s disease virus gene encoding pp38, an antigen associated with transformed cells. J Virol. 1991;65(12):6509–15. pmid:1658357
- 47. Liao Y, Lupiani B, Reddy SM. Latest Insights into unique open reading frames encoded by unique long (UL) and short (US) regions of Marek’s disease virus. Viruses. 2021;13(6). pmid:34070255
- 48. Becker Y, Asher Y, Tabor E, Davidson I, Malkinson M. Open reading frames in a 4556 nucleotide sequence within MDV-1 BamHI-D DNA fragment: evidence for splicing of mRNA from a new viral glycoprotein gene. Virus genes. 1994;8(1):55–69. pmid:8209423
- 49. Liu HC, Soderblom EJ, Goshe MB. A mass spectrometry-based proteomic approach to study Marek’s Disease Virus gene expression. Journal of virological methods. 2006;135(1):66–75. pmid:16563527
- 50.
Hunter G. Marek’s disease virus pathogenesis and latency. Master’s Thesis. Edinburgh, UK, University of Edinburgh, 2012.
- 51. Tischer BK, von Einem J, Kaufer B, Osterrieder N. Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. BioTechniques. 2006;40(2):191–7. pmid:16526409
- 52. Chang S, Ding Z, Dunn JR, Lee LF, Heidari M, Song J, et al. A comparative evaluation of the protective efficacy of rMd5deltaMeq and CVI988/ Rispens against a vv+ strain of Marek’s disease virus infection in a series of recombinant congenic strains of White Leghorn chickens. Avian Dis. 2011;55(3):384–90. pmid:22017035
- 53. Ning W, Jiang P, Guo Y, Wang C, Tan X, Zhang W, et al. GPS-Palm: a deep learning-based graphic presentation system for the prediction of S-palmitoylation sites in proteins. Briefings in bioinformatics. 2021;22(2):1836–47. pmid:32248222
- 54. Baines JD, Jacob RJ, Simmerman L, Roizman B. The herpes simplex virus 1 UL11 proteins are associated with cytoplasmic and nuclear membranes and with nuclear bodies of infected cells. J Virol. 1995;69(2):825–33. pmid:7815549
- 55. Nozawa N, Daikoku T, Koshizuka T, Yamauchi Y, Yoshikawa T, Nishiyama Y. Subcellular localization of herpes simplex virus type 1 UL51 protein and role of palmitoylation in Golgi apparatus targeting. J Virol. 2003;77(5):3204–16. pmid:12584344
- 56. Brignati MJ, Loomis JS, Wills JW, Courtney RJ. Membrane association of VP22, a herpes simplex virus type 1 tegument protein. J Virol. 2003;77(8):4888–98. pmid:12663795
- 57. Murphy MA, Bucks MA, O’Regan KJ, Courtney RJ. The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids. Virology. 2008;376(2):279–89. pmid:18452963
- 58. Daikoku T, Ikenoya K, Yamada H, Goshima F, Nishiyama Y. Identification and characterization of the herpes simplex virus type 1 UL51 gene product. The Journal of general virology. 1998;79 (Pt 12):3027–31. pmid:9880018
- 59. Loomis JS, Courtney RJ, Wills JW. Binding partners for the UL11 tegument protein of herpes simplex virus type 1. J Virol. 2003;77(21):11417–24. pmid:14557627
- 60. Loomis JS, Bowzard JB, Courtney RJ, Wills JW. Intracellular trafficking of the UL11 tegument protein of herpes simplex virus type 1. J Virol. 2001;75(24):12209–19. pmid:11711612
- 61. Maringer K, Stylianou J, Elliott G. A network of protein interactions around the herpes simplex virus tegument protein VP22. Journal of virology. 2012;86(23):12971–82. pmid:22993164
- 62. Hogue IB. Tegument Assembly, Secondary Envelopment and Exocytosis. Current issues in molecular biology. 2021;42:551–604. pmid:33622984
- 63. Sun A, Zhao X, Zhu X, Kong Z, Liao Y, Teng M, et al. Fully attenuated Meq and pp38 double gene deletion mutant virus confers superior immunological protection against highly virulent Marek’s disease virus infection. Microbiology spectrum. 2022;10(6):e0287122. pmid:36350141
- 64. Baaten BJ, Staines KA, Smith LP, Skinner H, Davison TF, Butter C. Early replication in pulmonary B cells after infection with Marek’s disease herpesvirus by the respiratory route. Viral immunology. 2009;22(6):431–44. pmid:19951180
- 65. Baigent SJ, Ross LJ, Davison TF. Differential susceptibility to Marek’s disease is associated with differences in number, but not phenotype or location, of pp38+ lymphocytes. The Journal of general virology. 1998;79 (Pt 11):2795–802. pmid:9820156
- 66. Calnek BW. Pathogenesis of Marek’s disease virus infection. Curr Top Microbiol Immunol. 2001;255:25–55. pmid:11217426
- 67. Burgess SC, Davison TF. Identification of the neoplastically transformed cells in Marek’s disease herpesvirus-induced lymphomas: recognition by the monoclonal antibody AV37. J Virol. 2002;76(14):7276–92. pmid:12072527
- 68. Chakraborty P, Vervelde L, Dalziel RG, Wasson PS, Nair V, Dutia BM, et al. Marek’s disease virus infection of phagocytes: a de novo in vitro infection model. The Journal of general virology. 2017;98(5):1080–8. pmid:28548038
- 69. Barrow AD, Burgess SC, Baigent SJ, Howes K, Nair VK. Infection of macrophages by a lymphotropic herpesvirus: a new tropism for Marek’s disease virus. The Journal of general virology. 2003;84(Pt 10):2635–45. pmid:13679597
- 70. Schermuly J, Greco A, Härtle S, Osterrieder N, Kaufer BB, Kaspers B. In vitro model for lytic replication, latency, and transformation of an oncogenic alphaherpesvirus. Proc Natl Acad Sci U S A. 2015;112(23):7279–84. pmid:26039998
- 71. Shek WR, Calnek BW, Schat KA, Chen CH. Characterization of Marek’s disease virus-infected lymphocytes: discrimination between cytolytically and latently infected cells. Journal of the National Cancer Institute. 1983;70(3):485–91 pmid:6300499
- 72. Bertzbach LD, van Haarlem DA, Härtle S, Kaufer BB, Jansen CA. Marek’s disease virus Infection of natural killer cells. Microorganisms. 2019;7(12). pmid:31757008
- 73. Bertzbach LD, Laparidou M, Härtle S, Etches RJ, Kaspers B, Schusser B, et al. Unraveling the role of B cells in the pathogenesis of an oncogenic avian herpesvirus. Proc Natl Acad Sci U S A. 2018;115(45):11603–7. pmid:30337483
- 74. Delecluse HJ, Hammerschmidt W. Status of Marek’s disease virus in established lymphoma cell lines: herpesvirus integration is common. J Virol. 1993;67(1):82–92. pmid:8380099
- 75. Kaufer BB, Jarosinski KW, Osterrieder N. Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation. The Journal of experimental medicine. 2011;208(3):605–15. pmid:21383055
- 76. Bowzard JB, Visalli RJ, Wilson CB, Loomis JS, Callahan EM, Courtney RJ, et al. Membrane targeting properties of a herpesvirus tegument protein-retrovirus Gag chimera. J Virol. 2000;74(18):8692–9. pmid:10954570
- 77. Crump C. Virus Assembly and Egress of HSV. Advances in experimental medicine and biology. 2018;1045:23–44. pmid:29896661
- 78.
Yasushi Kawaguchi YM, Hiroshi Kimura. Human herpesviruses. Singapore: Springer; 2018. 32–3 p.
- 79. Mettenleiter TC. Herpesvirus assembly and egress. J Virol. 2002;76(4):1537–47. pmid:11799148
- 80. Sucharita S, Krishnagopal A, van Drunen Littel-van den Hurk S. Comprehensive Analysis of the Tegument Proteins Involved in Capsid Transport and Virion Morphogenesis of Alpha, Beta and Gamma Herpesviruses. Viruses. 2023;15(10). pmid:37896835
- 81. Mettenleiter TC. Intriguing interplay between viral proteins during herpesvirus assembly or: the herpesvirus assembly puzzle. Veterinary microbiology. 2006;113(3–4):163–9. pmid:16330166
- 82. Chen Y, Li Y, Wu L. Protein S-palmitoylation modification: implications in tumor and tumor immune microenvironment. Frontiers in immunology. 2024;15:1337478. pmid:38415253
- 83. Dunn JR, Silva RF. Ability of Meq-deleted MDV vaccine candidates to adversely affect lymphoid organs and chicken weight gain. Avian Dis. 2012;56(3):494–500. pmid:23050465
- 84. Lee LF, Heidari M, Zhang H, Lupiani B, Reddy SM, Fadly A. Cell culture attenuation eliminates rMd5ΔMeq-induced bursal and thymic atrophy and renders the mutant virus as an effective and safe vaccine against Marek’s disease. Vaccine. 2012;30(34):5151–8. pmid:22687760
- 85. Hildebrandt E, Dunn JR, Cheng HH. The mut UL5-I682R Marek’s disease virus with a single nucleotide mutation within the helicase-primase subunit gene not only reduces virulence but also provides partial vaccinal protection against Marek’s disease. Avian Dis. 2015;59(1):94–7. pmid:26292541
- 86. Conrad SJ, Hearn CJ, Silva RF, Dunn JR. Codon deoptimization of UL54 in meq-deleted Marek’s disease vaccine candidate eliminates lymphoid atrophy but reduces vaccinal protection. Avian Dis. 2020;64(3):243–6. pmid:33205163
- 87. Sun A, Luo J, Wan B, Du Y, Wang X, Weng H, et al. Lorf9 deletion significantly eliminated lymphoid organ atrophy induced by meq-deleted very virulent Marek’s disease virus. Veterinary microbiology. 2019;235:164–9. pmid:31282374
- 88. Liao Y, Reddy SM, Khan OA, Sun A, Lupiani B. A novel effective and safe vaccine for prevention of Marek’s disease caused by infection with a very virulent plus (vv+) Marek’s disease virus. Vaccines. 2021;9(2). pmid:33669421
- 89. Conrad SJ, Oluwayinka EB, Heidari M, Mays JK, Dunn JR. Deletion of the viral thymidine kinase in a Meq-deleted recombinant Marek’s disease virus reduces lymphoid atrophy but Is Less protective. Microorganisms. 2021;10(1). pmid:35056456
- 90. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. Journal of molecular biology. 1967;26(2):365–9. pmid:4291934