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Journal of Virology, October 2007, p. 10575-10587, Vol. 81, No. 19
0022-538X/07/$08.00+0     doi:10.1128/JVI.01065-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Horizontal Transmission of Marek's Disease Virus Requires US2, the UL13 Protein Kinase, and gC{triangledown}

Keith W. Jarosinski,1* Neil G. Margulis,1 Jeremy P. Kamil,1 Stephen J. Spatz,2 Venugopal K. Nair,3 and Nikolaus Osterrieder1

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York,1 Southeast Poultry Research Laboratory, Agriculture Research Service, USDA, Athens, Georgia,2 Viral Oncogenesis Group, Institute for Animal Health, Compton, Berkshire, United Kingdom3

Received 17 May 2007/ Accepted 10 July 2007


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ABSTRACT
 
Marek's disease virus (MDV) causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated characterization of specific genes and genomic regions. The development of most MDV BACs, including pRB-1B-5, derived from a very virulent MDV strain, involved replacement of the US2 gene with mini-F vector sequences. However, when reconstituted viruses based on pRB-1B were used in pathogenicity studies, it was discovered that contact chickens housed together with experimentally infected chickens did not contract Marek's disease (MD), indicating a lack of horizontal transmission. Staining of feather follicle epithelial cells in the skins of infected chickens showed that virus was present but was unable to be released and/or infect susceptible chickens. Restoration of US2 and removal of mini-F sequences within viral RB-1B did not alter this characteristic, although in vivo viremia levels were increased significantly. Sequence analyses of pRB-1B revealed that the UL13, UL44, and US6 genes encoding the UL13 serine/threonine protein kinase, glycoprotein C (gC), and gD, respectively, harbored frameshift mutations. These mutations were repaired individually, or in combination, using two-step Red mutagenesis. Reconstituted viruses were tested for replication, MD incidence, and their abilities to horizontally spread to contact chickens. The experiments clearly showed that US2, UL13, and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.


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INTRODUCTION
 
Marek's disease virus (MDV), also known as Gallid herpesvirus 2, causes Marek's disease (MD), a lymphoproliferative disease in chickens characterized by the development of tumors in the viscera and other organs. The pathogenesis of infection with MDV can be divided into four phases (4). In the first phase, between 3 and 6 days postinfection (p.i.), the primary target cells of infection are bursa-derived (B) lymphocytes. Infection of activated CD4+ thymus-derived (T) lymphocytes follows in the second phase. During the second phase, viral replication typically decreases and a latent infection is established between 5 and 10 days p.i. in activated CD4+ T lymphocytes. The third phase is characterized by reactivation of MDV replication between 14 and 21 days p.i. and infection of feather follicle epithelium (FFE) cells. After the third phase, virus is shed from the chicken in dried FFE cells, and clinical MD symptoms and lymphomas may develop depending on the genetic susceptibility of the chickens and the virulence of the virus strain.

The MDV genome consists of the unique short (US) and long (UL) regions, flanked by the inverted repeat long (IRL) and short (IRS) regions, and the terminal repeat long (TRL) and short (TRS) regions. Through manipulation of the MDV genome using multiple approaches, specific genes or regulatory elements have been implicated in different stages of MDV pathogenesis such as tumor development and attenuation. MDV Eco Q (Meq or RLORF7), located within the RL regions, has been shown to be the prominent oncogene involved in tumor development (3, 18, 20), while the viral telomerase RNA (vTR), also encoded within the RL, plays a role in continued survival of transformed cells and metastasis (47). Attenuation of MDV, characterized by reduced lytic replication in vivo and MD incidence, has been attributed to a number of genes, but only a few MDV mutants and strains show what is considered "true" attenuation, which also includes increased virus replication in vitro (52). Only the RLORF4 and glycoprotein C (gC) (UL44) genes have been shown to satisfy these criteria by increasing viral replication and plaque sizes in vitro while decreasing viral replication and virulence in vivo (13, 45). Much of the work elucidating the role of specific genes or elements has used either the very virulent MDV strain Md5, whose genome had been cloned as a set of overlapping cosmid clones (40), or a bacterial artificial chromosome (BAC) clone of the very virulent RB-1B strain (pRB-1B-5), hereafter referred to as pRB-1B (38). One of the major drawbacks of pRB-1B was its inability to produce infectious virus that was able to horizontally spread from infected to uninfected chickens (2, 13). A number of possibilities existed to explain this finding, including the absence of US2, which has been shown to be nonessential for virus growth (36) and is replaced in the pRB-1B BAC clone by insertion of mini-F vector sequences (38). In addition to this possibility, single point mutations within a number of genes were identified following complete sequencing of the pRB-1B clone (44a). Of particular interest for the study of horizontal transmission of MDV, frameshift mutations were identified within the UL13, gC, and US6 (gD) genes that could potentially contribute to the inability of viruses reconstituted from pRB-1B (vRB-1B) to spread from chicken to chicken.

The MDV US2 orthologue is predicted to encode a protein with a molecular mass of ~30 kDa and has been shown to be nonessential for MDV replication in vitro and in vivo (36). The herpes simplex virus type 1 (HSV-1) US2 protein contains a hydrophobic N-terminal region and is believed to be membrane associated (23). Analysis of mutant viruses lacking US2 in HSV-1, pseudorabies virus (PRV), and equine herpesvirus 1 (EHV-1) have shown that it is nonessential for growth in vitro (8, 21, 24, 50), although US2-negative viruses showed slightly reduced replication characteristics (24, 50).

The orthologues of the serine/threonine protein kinase encoded by UL13 are conserved among all members of the Herpesviridae. The predicted UL13 protein of MDV has significant homology with those of HSV-1 and varicella-zoster virus (VZV), particularly at the site of the kinase domain. In HSV-1, UL13 is packaged in the tegument, phosphorylates viral protein 22 (VP22), infected-cell protein 22 (ICP22), ICP20, US3, and the viral Fc receptor (gE + gI) and also is capable of autophosphorylation (6, 17, 29, 32, 39). Likewise, the product of the VZV UL13 orthologue, open reading frame 47 (ORF47), is able to phosphorylate a number of viral and cellular proteins and, of particular relevance, has been shown to be required for efficient infection of T lymphocytes and skin in the SCID-hu mouse model (27). Little is known about the function of MDV UL13, but repair of the gene in pRB-1B did not restore the ability of reconstituted viruses to horizontally spread (2).

Homologues of gD are encoded within the US region of alphaherpesviruses. While gD is essential for almost all members of the virus subfamily analyzed thus far (7, 10, 16, 19, 51), VZV does not harbor gD (7) and gD of MDV is dispensable for virus growth in vitro, where it is silenced, and its expression seems to be restricted to FFE cells in vivo (31). Anderson et al. showed that insertion of a lacgpt cassette in place of gD did not alter in vitro or in vivo growth kinetics and did not prevent horizontal spread (1).

Alphaherpesvirus gC orthologues have multiple functions, both in vitro and in vivo. They play major roles in the primary attachment of cell-free virus to heparin- and chondroitin-like glycosaminoglycans on the surface of cells (25, 41), and involvement of gC in a late step of virus egress from cultured cells has been shown for PRV and EHV-1 (25, 34). In the case of VZV, gC is a major determinant for virulence in skin as demonstrated in the SCID-hu mouse model (26). It has been previously hypothesized that gC is important for virus shedding and transmission to uninfected chickens (28); however, this could not be confirmed due to the lack of a revertant virus in this study.

In this report, the contributions of US2, UL13, gC, and gD in MDV replication, tumorigenesis, and horizontal spread were examined by restoring US2 and repairing the ORFs encoding the UL13 protein kinase, gC, and gD individually or in various combinations. Our comprehensive experiments allow us to conclude that horizontal spread of recombinant pRB-1B-derived viruses minimally required restoration of US2, gC, and UL13 in combination.


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MATERIALS AND METHODS
 
Cell cultures and viruses. Chicken embryo cell (CEC) cultures were prepared from 10-day-old specific-pathogen-free (SPF) embryos following standard methods (43) and used to reconstitute viruses from pRB-1B DNA. Chicken kidney cell (CKC) cultures were prepared from 14-day-old SPF chickens (43) and used to propagate pRB-1B-derived viruses and wild-type MDV strain RB-1B (42), kindly provided by Karel A. Schat (Cornell University, Ithaca, NY). All reconstituted BACs were used at ≤5 passages, while RB-1B was used at passage 12.

Recombinant viruses were reconstituted by transfecting CEC cultures with purified BAC DNA using the CaPO4 precipitation method (44) with or without pCAGGS-NLS/Cre, a plasmid expressing the Cre enzyme for excision of mini-F sequences using loxP sites (a gift from Michael I. Kotlikoff, Cornell University, Ithaca, NY). For most pRB-1B-derived viruses, infected CEC cultures were passaged onto CKC cultures after the development of plaques following transfection and expanded. Recombinant virus stocks were examined for the removal of mini-F vector sequences using specific primers described below.

Restoration of US2 in pRB-1B. To restore US2 in the pRB-1B BAC, a RecA-based shuttle mutagenesis strategy was employed as previously described (15). To provide one of the flanks for RecA-mediated recombination, pHA1 (44) was digested with SpeI and EcoRI, and the 1,020-bp fragment encompassing a loxP site, sopC/parA, and part of sopB was ligated into pSP72 (Promega, Inc., Madison, WI), which had been linearized with EcoRI and XbaI (compatible end with SpeI), resulting in pSP72-SopB.C. For the other flank, primers that amplify the US2 and part of the US3 region from RB-1B were designed to engineer EcoRI and BamHI sites on the ends and were as follows (restriction site underlined): US3_US2 BamHI F, 5'-GGATCCAACGGATGGACTTGCAGGCA-3', and US3_US2 EcoRI R, 5'-GAATTCTAATGACTACCGGCTCTAC-3'. The amplified PCR product was cloned into the TOPO TA cloning kit pCR2.1 vector (Invitrogen Corp., Carlsbad, CA) and sequenced to verify that no mutations were introduced during the amplification reaction. In order to produce the final shuttle vector for restoring US2, the US2/US3 flanking sequence was released from the TOPO vector using the EcoRI restriction enzyme and ligated into EcoRI-linearized pSP72-SopB.C to generate pSP72-SopB.C/Us3.2. Finally, the SphI-BamHI fragment from pSP72-SopB.C/Us3.2 was ligated into pST76K-SR, creating the shuttle plasmid pST76K-SR/SopB.C/Us3.2 using compatible ends. The final US2 restoration shuttle mutagenesis vector was used to obtain cointegrates in pRB-1B. During resolution of the cointegrates (15), two possible recombination events were possible and the correct, US2-restored pRB-1B clones were identified by Southern blot analysis and further characterized by sequencing.

Repairing UL13, gC, and gD in pRB-1B. Primers used for two-step Red-mediated recombination are listed in Table 1. Mutagenesis was performed as previously described (46). Briefly, amplification reactions were performed using Reddy Master Mix (ABgene, Rochester, NY) containing 0.2 mM deoxynucleoside triphosphates, 1.5 mM MgCl2, 20 mM (NH4)2SO4, 75 mM Tris-HCl (pH 8.8), 0.01% (vol/vol) Tween 20, 1.25 U Thermoprime Plus DNA polymerase, ~100 ng pEPkan-S plasmid, and 0.2 mM of the forward and reverse primers. The PCR product was purified from an agarose gel using the QIAquick gel extraction kit (QIAGEN, Inc.) following the manufacturer's protocol. The purified product was eluted in 30 µl nuclease-free water, and 2 to 5 µl was used for electroporation into recombination-competent GS1783 Escherichia coli cells (a kind gift from Gregory A. Smith, Northwestern University, Chicago, IL) containing pRB-1B. Following electroporation, bacteria were incubated at 32°C with shaking for ~1 h and then plated onto Luria-Bertani (LB) agar containing 30 µg/ml chloramphenicol and 50 µg/ml kanamycin. Kanamycin-resistant clones were then examined for insertion of Kanr into the respective loci using Southern blot analysis. BamHI and HindIII were both used to examine clones for spurious changes in restriction fragment patterns.


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  		TABLE 1. Primers used for repairing MDV genes in pRB-1B

Following confirmation of insertion of Kanr into the correct locus for each respective mutagenesis reaction, Kanr was excised by first expressing the I-SceI restriction enzyme in GS1783 E. coli cells through induction with arabinose, followed by induction of the Red recombination machinery through a rise in temperature. Briefly, 100 µl of an overnight culture of GS1783 E. coli cells containing the BAC grown in LB broth plus chloramphenicol and kanamycin was inoculated into 2 ml of LB broth containing only chloramphenicol. Bacteria were incubated at 32°C for 2 to 4 h with shaking, followed by addition of 2% (wt/vol) L-arabinose (Sigma, St. Louis, MO) to the culture at a 1:1 ratio, and incubated for another 1 h at 32°C. Finally, cells were incubated at 42°C for 30 min. The culture was then shaken at 32°C for another 1 to 2 h, and 100 µl of 10–1 to 10–6 dilutions of the culture was plated onto LB agar plates containing only chloramphenicol. After 24 to 48 h, individual colonies were replica plated onto LB containing chloramphenicol or LB containing chloramphenicol plus kanamycin. Kanamycin-susceptible clones were further screened by Southern blot analyses, followed by nucleotide sequencing for confirmation purposes.

In those pRB-1B clones where multiple genes were repaired, the order in which genes were restored is as follows: 1216 and 1217 (UL13 repaired) and 1218 (gC repaired) were generated from the 1194 (US2-restored) clone, 1231 and 1232 were generated by repairing gC in 1217 (US2 restored and UL13 repaired), and 1272 was generated by repairing gD in 1232 (US2 restored and UL13 + gC repaired). Clones 1265 (gD repaired) and 1355 (gC repaired) were generated from 1193. All clones and their genotypes are given in Table 2.


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  		TABLE 2. Designation of virus by number

Southern blot analysis. Probes used in Southern blot analysis were prepared using either the DIG-High Prime (MDV) or PCR DIG Probe Synthesis (Kanr) kit from Roche Applied Science according to the manufacturer's protocols as described earlier (13). For hybridization, DNA was separated by 0.8% agarose gel electrophoresis, gels were stained with ethidium bromide, and DNA was transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) according to the manufacturer's instructions.

DNA extraction from blood cells. To measure viral DNA copies in chicken blood using quantitative PCR (qPCR) assays, DNA was extracted from whole blood as follows. Forty microliters of blood was obtained from the wing vein, mixed with 20 µl 0.1 M EDTA, and then frozen at –80°C until all samples were collected. Ten microliters of blood-EDTA mix was used to collect DNA using the DNeasy 96 tissue kit from QIAGEN, Inc. (Valencia, CA), according to the manufacturer's instructions. Final DNA preparations were eluted in 100 to 200 µl elution buffer heated to 70°C.

qPCR assays. Quantification of MDV genomic copies using qPCR was performed as previously described (13, 53). Briefly, primers and probe specific for the MDV ICP4 were used in qPCR assays. DNA loading for each sample was normalized using primers and a probe specific for the chicken inducible nitric oxide synthase (iNOS) gene.

For the generation of standard curves in qPCR assays, a plasmid containing iNOS and 1232 DNA (Table 2) were used. Serial 10-fold dilutions of each respective plasmid or BAC were used for generating standard curves, starting with approximately 500 pg of DNA. Total copy numbers were determined using the following formula: [(pg of input)(1 pmol/340 pg)(1/template size in bp)(1 mol/1 x 1012 pmol)]/(6.02 x 1023 copies/mol), as previously described (14), and standard curves were generated by plotting the threshold cycle value at each dilution with the total copies. The coefficient of regression was always >0.99 for standard curves.

All qPCR assays were performed in an ABI Prism 7500 Fast Real-Time PCR System (Applied Biosystems, Inc.), and the results were analyzed using Sequence Detection Systems version 1.4 software. Each qPCR mixture contained TaqMan Fast Universal Master Mix, No AmpErase UNG (Applied Biosystems, Inc.), 9.5 µl DNA, 25 pmol of each gene-specific primer, and 10 pmol of the gene-specific probe in a 20-µl volume. Thermal cycling conditions were as follows: 95°C for 20 s, followed by 40 cycles at 95°C for 3 s and 60°C for 30 s. Using the standard curve generated for each gene, the numbers of copies for ICP4 and iNOS were determined by using the threshold cycle value for that sample as previously described (13).

In vivo experiments. SPF P2a (major histocompatibility complex: B19B19) chickens were obtained from departmental flocks and housed in isolation units. Water and food were provided ad libitum. All experimental procedures were conducted in compliance with approved Institutional Animal Care and Use Committee protocols. Chickens were inoculated intra-abdominally with 500 to 2,000 PFU of the various viruses at 1 day of age. Chickens were evaluated daily for symptoms of MD, euthanized, and examined for gross MD lesions when birds showed clinical evidence of MD. In most reconstituted viruses, mini-F vector sequences were removed by Cre excision, unless otherwise noted as "+F." Chickens were assigned to treatment groups using a randomized table.

Statistical analysis. Significant differences in means for qPCR assays were determined using either Student's t test or the Tukey-Kramer comparison of means.


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RESULTS
 
Restoration of US2 and removal of mini-F sequences in viruses reconstituted from pRB-1B. US2 has been shown to be nonessential for MDV replication in vitro and in vivo (36). It has also been shown, however, that vRB-1B reconstituted from pRB-1B (v1193) was unable to spread from chicken to chicken (2, 13). This led us to hypothesize that, although US2 is dispensable for MDV replication, it may be important for virus shedding and spread to sentinel chickens. It is also possible that the presence of mini-F vector sequences in the reconstituted virus may alter the recombinant virus's phenotype.

To address these questions, we restored US2 in the pRB-1B clone and used Cre-loxP recombination to remove mini-F vector sequences from reconstituted BAC viruses (Table 2) during transfection. Primers were designed to flank mini-F sequences, and PCR assays were performed on DNA extracted from infected-cell cultures. Figure 1 shows a schematic representation of the genomic region and the expected amplicons using primer pair SORF3vCreF5-Us2vCre B8 or SopC F-US3-SeqUS2 AS (Table 3) in the presence or absence of restored US2 and/or removal of mini-F vector sequences (Fig. 1A to C). PCR analyses of DNA collected from CKC cultures infected with the various viruses revealed that the original v1193 reconstituted from the original pRB-1B infectious clone (v1193), as well as v1265, in which only gD had been repaired, did not yield a product of 714 bp (Fig. 1D). PCR analyses of all viruses with restored US2 and wild-type RB-1B resulted in the expected amplification of a 714-bp product, although the product appeared to be slightly smaller in the case of wild-type RB-1B. This was likely caused by the absence of the loxP sites in the parental virus that still remain in the BAC-derived viruses. The SopC F primer binds to sequences within mini-F sequences; therefore, viral DNA with mini-F sequences removed would not yield an amplification product, while viral DNA containing mini-F sequences would result in products of either 1.5 kb (+US2) or 988 bp ({Delta}US2) using primer pair SopC F-US3-SeqUS2 AS (Fig. 1A and B). This was confirmed with purified BAC DNA and viral DNA when Cre was not added. Taken together, the PCR analyses revealed complete removal of mini-F vector sequences by cotransfection of a Cre-expressing plasmid after at least two passages in CEC and/or CKC cultures.


Figure 1
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  		FIG. 1. Analysis of US2 restoration by PCR. Shown is a schematic representation of the expected PCR product sizes using the SORF3vCre F5 (F5) and US2vCre B8 (B8) or SopC F and Us3-SeqUs2 AS (AS) primers and diagnostic PCR assays for insertion of US2 and removal of mini-F vector sequences from reconstituted viruses. (A to C) The expected PCR products using the two primer sets in reconstituted viruses lacking US2 and containing mini-F sequences (A), containing restored US2 and mini-F sequences (B), or containing restored US2 but lacking mini-F sequences after Cre excision (C) are shown. Using primers SopC F and AS, a 1,508- or 988-bp fragment is amplified in the presence or absence of US2, respectively, if mini-F sequences are present. No product will be amplified if mini-F sequences are removed, since the SopC F primer binds within the mini-F vector sequences. Using the F5 and B8 primers, a 714-bp fragment is amplified in US2-restored viruses lacking mini-F sequences. If vector sequences are present, the primers would be able to bind their respective complementary sequences but would not be amplified using our PCR parameters. In the absence of US2, the B8 primers would not bind. (D) The top panel shows PCR assays for the restoration of the US2 gene in reconstituted viruses (v). BAC DNA was transfected into CEC cultures with (+) or without (–) the pCAGGS-NLS/Cre plasmid, and reconstituted viruses were passaged in CKC cultures up to five times. DNA was extracted from infected CKC cultures and used in PCRs using previously described methods (33). Amplification of US2 was evident in each virus in which US2 was restored. The bottom panel shows PCR assays for the removal of mini-F sequences when viruses were reconstituted with the Cre enzyme present. The asterisks indicate BAC DNA used as controls.


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  		TABLE 3. Primers used for sequencing and diagnostics

Increased viral replication of recombinant MDV after removal of mini-F vector sequences. We next tested the reconstituted viruses in chickens (experiment 1). Fifteen chickens per group were inoculated with 1,000 PFU of v1193+F, v1193, v1194, or wild-type RB-1B (Table 2) in separate isolation units. Blood was collected at 4, 7, 14, 21, and 28 days p.i. and used for analysis of viral replication by qPCR. In addition to inoculated chickens, an additional five chickens were left uninfected for each group as contact controls. The experiment was terminated at 63 days p.i., when all chickens, including contact chickens, were examined for gross MD lesions. Blood was collected from contact chickens for use in qPCR assays. Removal of mini-F vector sequences in reconstituted viruses increased virus replication and MD incidence (Fig. 2A). However, the restoration of US2 did not affect virus replication as measured by viremia levels or MD incidence (Fig. 2B). When MDV genomic copies were measured in peripheral blood, wild-type RB-1B had significantly higher levels at 7 to 21 days p.i. than did the vRB-1B viruses, consistent with our previous results (13). Restoration of US2 did not affect the level of replication as evidenced by virtually identical numbers of MDV genomes after infection with the v1193 ({Delta}US2) or v1194 (+US2) virus. It appeared, however, that removing mini-F vector sequences increased viral replication in vivo, which was supported by lower MDV copy numbers in peripheral blood in chickens infected with v1193+F than in the blood of those in the v1193-infected group. Removal of mini-F sequences also appeared to increase MD incidence (Fig. 2B), although statistical analyses were not performed with these small sample sizes.


Figure 2
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  		FIG. 2. In vivo replication and MD incidence in chickens infected with US2-restored vRB-1B with (+F) or without mini-F sequences. (A) Mean MDV genomic copies/1 x 106 blood cells ± standard errors of the means using qPCR assays. Values for each group that were significantly higher ({wedge}) or lower (*) than those of all other groups (P < 0.05) are indicated. (B) Percent MD incidence for each group during the 9-week evaluation period.

During the course of experiment 1, sentinel chickens housed together with chickens with wild-type RB-1B virus developed MD, and at the termination of experiment 1 (63 days p.i.) all sentinel chickens had developed lymphomas (10/10). In contrast, none of the contact chickens in all other groups developed MD. In addition, no viral DNA could be detected in any group except the RB-1B group in blood DNA recovered from sentinel chickens (data not shown).

As previously shown (13), the level of viral replication in chickens infected with pRB-1B-derived viruses is generally 10- to 100-fold lower than that for chickens infected with wild-type RB-1B (Fig. 2A). We hypothesized that a lower level of virus replication, possibly in conjunction with a more advanced immune competence (sentinel chickens were at least 2 weeks of age before being exposed to virus shed by the inoculated birds), may reduce the likelihood of virus spread to sentinel chickens, a phenomenon that is often seen with attenuated strains. We conducted an additional experiment to examine if younger chickens could become horizontally infected (experiment 2), in which 10 1-day-old chickens per group were inoculated with 2,000 PFU of v1193+F, v1193, or v1194 (Table 2). At 2 and 3 weeks p.i., five additional 1-day-old sentinel chickens were added to each group. The results in experiment 2 corroborated those of experiment 1, and no spread to sentinel chickens was observed (data not shown). The results of these experiments, therefore, led us to conclude that restoration of US2 did not increase virus replication levels in vivo and was not sufficient to confer the ability of horizontal spread on pRB-1B-derived viruses, even in the absence of mini-F vector sequences.

Since spread of vRB-1B could not be seen, the question was raised as to whether the virus is able to infect the FFE cells. Skin tissues were collected from infected and uninfected chickens and stained for MDV antigens using immunohistochemical techniques. In agreement with a recent publication (2), MDV antigens could be readily detected in the skin FFE cells in chickens infected with all viruses examined (data not shown).

Frameshift mutations in genes potentially involved in MDV horizontal transmission. Following complete sequence analysis of the pRB-1B clone (1193), it was found that multiple changes were seen compared to the published Md5 strain (48). Of particular interest were genes which contained frameshift mutations and which we surmised to be potentially important for horizontal transmission. Among them were the genes encoding gC, gD, and the UL13 serine/threonine protein kinase. The frameshift mutations resulted from an additional A in a stretch of six A's located 508 bp downstream of the UL13 start codon (Fig. 3A), an additional T in a stretch of eight T's 46 bp downstream of the gC start codon (Fig. 3B), and an additional T in a stretch of eight T's 26 bp downstream of the gD start codon (Fig. 3C) with all resulting in early termination of predicted protein sequences. The frameshift in UL13 led to termination of translation at 176 amino acids as previously described (2) lacking the serine/threonine protein kinase activation site (underlined in Fig. 3A). Using vector NTI software, a potential ORF was predicted after the premature stop codon in the 1193 sequence that encodes the serine/threonine protein kinase activation site, but it is unknown whether this ORF is actually translated and could be functionally active. The frameshift mutation within gC leads to a truncated protein of only 30 amino acids. Again, there is a potential ORF downstream of the frameshift mutation that could produce an N-terminally truncated gC protein containing the transmembrane domain (underlined in Fig. 3B). The mutation identified in gD leads to early termination of translation at 11 amino acids (Fig. 3C). It has been predicted that the second ATG codon is required for a synthesis of full, mature glycosylated gD (54), which would be unaffected by the additional T in the 1193 gD ORF. Translation initiation at the first codon would include a transmembrane domain at the N terminus that would be excluded if the second ATG codon were used for translation initiation, while both proteins would include a transmembrane domain from positions 358 to 380 of the long protein or 340 to 362 of the shorter protein.


Figure 3
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  		FIG. 3. Frameshift mutations within UL13, gC, and gD in pRB-1B and their predicted protein sequences. The nucleotide sequences of UL13, gC, and gD in which frameshift mutations were identified are shown in panels A, B, and C, respectively, compared to the published Md5 sequence (48). Numbers indicate the respective nucleotide, and bold capitalized letters indicate the additional nucleotide causing the frameshift. Predicted protein sequences for each gene are compared to the Md5 sequence. Potential translation initiation sites are indicated with a bold M. Truncated proteins are shown for UL13, gC, and gD. Underlined amino acid sequences indicate the serine/threonine protein kinase active site for UL13 (A) and transmembrane domains for gC (B) and gD (C).

Repair of frameshift mutations of UL13, gC, and gD. The respective genes were repaired using two-step Red mutagenesis, and wild-type sequences were restored in the various recombinant genomes individually or in various combinations. Table 2 gives a synopsis of the engineered genomes and viruses.

Since there are no antibodies available for the US2 and UL13 proteins, and since gD is not expressed in vitro (54), we were unable to examine if protein expression was restored in the reconstituted viruses. However, we were able to confirm gC protein expression using the A6 antibody in Western blot analyses of infected CKC culture supernatants through enrichment by precipitation using concanavalin A-coated Sepharose beads (45). It was clearly shown that gC was expressed in v1218, v1232, v1355, and v1272 (not shown), in addition to wild-type RB-1B, which served as a positive control, while v1193 (not shown), v1194, and v1216 had no detectable gC protein in infected-cell supernatants (Fig. 4).


Figure 4
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  		FIG. 4. Western blot analysis of secreted gC in repaired viruses. Glycoproteins of uninfected or MDV-infected CKC cultures were precipitated with concanavalin A-Sepharose beads and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by detection of gC using the gC-specific monoclonal antibody A6 as previously described (45). The gC protein was detected only in gC-repaired viruses with the expected sizes of 57 to 65 kDa, while uninfected and 1194- or 1216-infected CKC cultures were negative.

In vivo replication and MD incidence of viruses with repaired genes. Two animal trials were performed with the various restored and repaired viruses to evaluate the contributions of the mutated genes to replication and MD incidence (experiment 3). In trial 1, chickens were inoculated with 500 PFU of v1193, v1194, v1216, v1231, or v1232 (Table 2), while in trial 2, chickens were inoculated with 1,000 PFU of v1193, v1216, v1218, v1265, v1232, v1272, or wild-type RB-1B (Table 2). At 3, 7, 10, 14, and 21 days p.i., wing vein blood was collected from 10 (trial 1) or 8 (trial 2) chickens per group and DNA was extracted for use in qPCR assays to measure viral replication. MD incidence was evaluated up to 51 (trial 1) and 37 (trial 2) days p.i., at which time blood was also collected from all surviving chickens.

In the first trial, replication of the repaired viruses was approximately 10-fold higher than that of v1193 (Fig. 5A). In the second trial, wild-type RB-1B virus was included for comparison with the recombinants, in particular the fully restored virus (v1272). Again, the general trend appeared to be that each of the repaired viruses had enhanced replication, although none reached the levels of RB-1B (Fig. 5C).


Figure 5
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  		FIG. 5. In vivo replication and MD incidence in chickens infected with repaired vRB-1B. (A) Mean MDV genomic copies/1 x 106 blood cells ± standard errors of the means (error bars) using qPCR assays for trial 1 with v1193, v1194, v1216, v1231, or v1232 (Table 2). Values for each group that were significantly higher ({wedge}) or lower (*) than those of all other groups without the same annotation (P < 0.05) are indicated. (B) Percent MD incidence for each group during the 52-day evaluation period in trial 1. (C) Same as in panel A for trial 1 except that v1193, v1216, v1218, v1265, v1232, v1272 (Table 2), or wild-type RB-1B was used. (D) Same as in panel B for trial 1 during an evaluation period of 37 days.

Consistent with earlier findings, there was little difference in overall MD incidence between viruses in trial 1, with all viruses inducing MD incidences of >80% up to 56 days p.i. (Fig. 5B). In trial 2, the experiment was terminated at 37 days p.i., at which time point all viruses, except the original vRB-1B (v1193), led to >80% MD incidences; however, the rate at which MD developed in chickens infected with the RB-1B virus was greater, with 89% of chickens developing clinical MD by 4 weeks p.i., while only up to 50% of chickens infected with the recombinant viruses had clinical MD at that time point after infection (Fig. 5D).

Horizontal spread of vRB-1B with repaired genes. To test for the ability of restored viruses to horizontally spread to uninfected chickens, 10 1-day-old chickens per group were inoculated with 1,000 PFU of the v1194, v1216, v1217, v1218, v1231, or v1232 virus (Table 2) and housed in separate isolation units (experiment 4). At 2 and 3 weeks p.i., five additional 1-day-old chickens were added to each group. Figure 6A shows MD incidences for inoculated chickens in each group. At the termination of the experiment at 7 weeks p.i., all chickens were examined for gross MD lesions, and we discovered that sentinel chickens housed with those infected with the v1231 (4/10) and v1232 (6/10) viruses, in which US2 was restored and UL13 and gC were repaired to wild-type sequences, contained gross lesions. In contrast, none of the sentinel chickens placed with any of the other groups contained gross MD lesions. Using qPCR assays on DNA from blood collected from all sentinel chickens in all groups revealed that, in perfect agreement with the postmortem examinations, no genomic MDV DNA could be detected in sentinel chickens housed with birds infected with v1194, v1216, v1217, or v1218. In contrast, 4/9 and 9/9 contact chickens had detectable levels of MDV DNA in their bloodstream when they were housed with animals inoculated with either the v1231 or the v1232 virus, respectively.


Figure 6
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  		FIG. 6. Incidence of MD in inoculated or contact chickens with repaired vRB-1B viruses. (A) The top panel shows the MD incidence of chickens inoculated with v1194, v1216, v1217, v1218, v1231, or v1232 over a 7-week period. Below is a table showing the number of chickens positive for gross lesions or amplification of MDV genomic copies using qPCR assays at the termination of the experiment. Chickens that died prior to the termination of the experiment due to MD were not used for blood DNA collection; thus, the total number of chickens sampled for qPCR assays may vary from the total number of chickens evaluated for gross lesions. The asterisk (*) denotes that chickens were excluded because the control qPCR (iNOS amplification) was undetectable, and thus they were excluded from the group for qPCR assays. (B) Same as in panel A, except that the experiment was extended to 13 weeks p.i. and used v1193, v1216, v1218, v1265, v1232, v1272, or wild-type RB-1B.

Requirement of both UL13 and gC for horizontal spread of MDV. It had previously been shown that deletion of gC in the RB-1B strain abolished spread (28); however, in this report, it could not be concluded that this property was completely due to the loss of the ability of the virus to spread and not due to a defect in virus replication in infected chickens. Since the two independent viruses (v1231 and v1232), in which US2 was restored and UL13 and gC were repaired, were able to spread to uninfected chickens, we tested the individual contribution of gC alone for spread. It has recently been shown that repairing UL13 alone in pRB-1B did not restore horizontal spread in reconstituted viruses (2).

In the last animal experiment (experiment 4), we tested v1216, v1218, and v1232 and compared their properties to three newly developed viruses, in which gC and gD were repaired but which still lacked US2, as well as v1272 (US2 restored and UL13, gC, and gD repaired), for an extended period of time. Chickens were inoculated with 2,000 PFU of the following viruses: v1193 (n = 9), v1194 (n = 7), v1216 (n = 9), v1218 (n = 9), v1355 (n = 10), v1265 (n = 9), v1232 (n = 9), and v1272 (n = 10). Each group was placed in separate isolation units with 10 to 20 uninfected sentinel chickens at the time of inoculation. The number of birds in groups without evidence of MD was reduced at 9 weeks p.i. to provide the required space/bird area. The remaining chickens were kept until 13 weeks p.i., when the experiment was terminated.

Figure 6B shows MD incidences of chickens inoculated with the various viruses as well as the MD incidences in sentinel contact chickens housed with the inoculated birds. Consistent with earlier results, we again found that the v1232 virus (Table 2) was able to spread horizontally, with 14 out of 18 chickens developing MD. A similar efficiency for spread was observed for the v1272 virus with 14 out of 16 contact chickens developing MD. Interestingly, and in contrast to earlier findings in experiment 4, two chickens housed with birds inoculated with the v1218 virus, with US2 restored and gC repaired, developed MD in the 7th and 8th weeks of the experiment. By the termination of the experiment, a total of six contact chickens in the v1218 group developed MD. In order to ensure that virus did not spread in the remaining groups, blood DNA was collected from all chickens euthanized at 9 and 13 weeks and tested using qPCR assays. As expected, sentinel chickens in the v1193, v1194, v1216, v1265, and v1355 groups remained negative for MDV DNA, and horizontal transmission in these groups could be excluded.

Natural restoration of UL13 in gC-repaired virus leads to spread. Although contact animals in the v1218 group developed MD, it was apparent that this spread occurred only at very late times after infection. We therefore determined the nucleotide sequences of the viral UL13, gC, and gD genes from DNA collected from liver and kidney tumors isolated from these chickens using the primers shown in Table 3. The sequencing analysis showed that UL13 had naturally reverted to the intact sequence reported for Md5. To exclude potential mix-ups in the inoculates, we also sequenced each gene from the virus stocks that were originally used for the infection of chickens, and we were able to confirm that, in the v1218 virus stock, UL13 contained the frameshift mutation disrupting the ORF, while gC was repaired as intended (data not shown).


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DISCUSSION
 
The development of recombinant DNA technology for MDV by cloning of the viral genomes as a set of overlapping cosmids (40) or in their entirety as single, infectious BACs (30, 37, 38, 44) has contributed considerably to the understanding of MDV pathogenesis over the past few years. Recombinant virus reconstituted from pRB-1B was shown to be highly oncogenic, generally causing tumors in >90% of susceptible chickens by 8 weeks p.i. (38); however, vRB-1B, unlike its parent RB-1B, was shown not to be transmitted horizontally to sentinel chickens (2, 13). We here report that two frameshift mutations in the infectious pRB-1B genome, the insertion of an additional T nucleotide in a stretch of eight T's in gC and an additional A in a stretch of six A's in UL13, are responsible for the failure of vRB-1B to spread horizontally. Repair of both ORFs in pRB-1B, followed by reconstitution, resulted in vRB-1B that is fully capable of spreading to contact chickens.

Little is known about the mechanism of MDV horizontal transmission. MDV replicates in vivo by initially infecting B lymphocytes, followed by infection of activated T lymphocytes, and finally, the virus reaches FFE cells where infectious virus is shed from the skin. MDV stays infectious for extended periods of time in dust and dander collected from chicken houses, and ultimately, naïve animals inhale dander containing the infectious virus to complete the infectious cycle (35). It is assumed that pulmonary macrophages take up infectious virus by phagocytosis of infectious skin cell debris, whereupon primary infection is established. It is unknown what changes occur in virus replication and assembly to allow production of cell-free, infectious virus, ultimately resulting in horizontal transmission. The fact that the vRB-1B reconstituted from pRB-1B induces tumors and reaches FFE cells, where it appears to go through a complete replicative cycle as evidenced by the production of late genes detected with antibodies against early and late viral proteins (2), suggests that its inability to spread was a defect in the composition of virus produced in FFE cells. We therefore assume that the defect of vRB-1B in horizontal virus spread is caused either by an inefficient or altered assembly process that would preclude the release of infectious virus or by inefficient virus entry or uptake into cells of the naïve chicken, most likely mucosal epithelia and/or phagocytic cells of the upper and lower respiratory tract.

MDV is a unique alphaherpesvirus, with respect to its pathogenesis and the fact that it can cause tumors, as well as its requirement for membrane and tegument (glyco-) proteins in virus assembly, maturation, and cell-to-cell spread. Concerning the requirements for tegument and membrane proteins, MDV is most closely related to VZV (35). For example, VZV and MDV propagation in vitro does not require the product of UL48, viral protein 16 (VP16), which is important for efficient growth of other members of the virus subfamily such as HSV-1, EHV-1, and PRV (5, 11, 49). In contrast, VP22 encoded by UL49 is essential for propagation of MDV and VZV but not for HSV-1, EHV-1, or PRV (9, 12). Likewise, gE and gI are essential for both VZV and MDV, while they are dispensable for growth of other, related Alphaherpesvirinae. gD homologues are encoded within the US region of alphaherpesviruses, together with gE, gI, and gG (absent in VZV and MDV), and most likely result from sequence duplication early in the evolution of the virus subfamily (22). While gD is essential for most members of the virus subfamily analyzed thus far (7, 10, 16, 19, 51), gD is dispensable for virus growth in vitro in the case of MDV, where it is silenced and its expression appears to be restricted to FFE cells in vivo (31). VZV does not harbor gD (7). Anderson et al. showed that insertion of a lacgpt cassette in place of the gD ORF did not prevent horizontal spread (1). Repair of the gD ORF in pRB-1B to the consensus Md5 sequence did not affect virus pathogenesis or transmission. Following this study, we retrospectively compared the DNA sequence of gD from the original RB-1B isolated in 1982, which showed that it also contained an additional T at position 26 of the predicted ORF, identical to what we initially found in pRB-1B (K. W. Jarosinski, unpublished observation). Thus, the major question raised from this information is if gD has a function in MDV replication or pathogenesis at all. It is conceivable that the function of gD has become completely irrelevant for VZV and MDV pathogenesis over time or that the gene duplication event was "incomplete," at least in the case of VZV. This latter interpretation may be supported by the fact that both MDV and VZV do not encode a gG orthologue; however, the predicted protein sequence of MDV gD appears to contain gG-like motifs (K. W. Jarosinski, unpublished observation).

It has been previously hypothesized that gC is important for virus shedding and transmission to uninfected chickens (28). This interpretation was based on the analysis of a gC-negative mutant of RB-1B, which was unable to spread from animal to animal; however, the level of infection with the virus was found to be severely impaired, and the lack of a revertant virus made it very difficult to truly appraise the role of gC in horizontal transmission. Alphaherpesvirus gC orthologues have multiple functions, both in vitro and in vivo. Most have a major role in the primary attachment of cell-free virus to heparin- and chondroitin-like glycosaminoglycans on the surface of cells, but involvement of gC in a late step of virus egress from cultured cells has been shown for PRV and EHV-1 (25, 41). In the case of VZV, gC is a major determinant for virulence in skin as demonstrated in the SCID-hu mouse model (26). The data presented in this report show that a frameshift in the gC ORF results in a virus that is able to cause tumors but is defective in horizontal transmission. Repair of the ORF to the wild-type sequence, individually or in combination with US2, did not restore the virus's ability to infect sentinel chickens. However, restoration of US2 and repair of gC together with UL13 resulted in a virus that was capable of being transmitted horizontally.

With regard to the UL13 protein, it is worthwhile to note that the ORF was repaired recently in pRB-1B individually (2). It was shown that, similarly to the situation described for gC, the kinase is not sufficient to restore horizontal transmission to pRB-1B-derived viruses. The UL13 serine/threonine kinase is conserved among all members of the Herpesviridae. The predicted UL13 protein of MDV has high homology with the HSV-1 and VZV UL13 orthologues, in particularly at the site of the kinase domain (amino acids 264 to 276 of MDV). In HSV-1, UL13 is packaged in the tegument; phosphorylates VP22, ICP22, ICP20, US3, and the viral Fc receptor (gE + gI); and also is capable of autophosphorylation (6, 17, 29, 32, 39). Likewise, the product of the VZV UL13 orthologue, ORF47, is able to phosphorylate a number of viral and cellular proteins and, of particular relevance, has been shown to be required for efficient infection of T lymphocytes and skin in the SCID-hu mouse model (27). To date, little is known about the function of the MDV UL13, but the mutation identified and repaired here together with previous findings (2) indicates that UL13 is not required for efficient virus replication in vitro or lymphocytes in vivo. Thus, the only essential role that it may play is during final assembly of virus in FFE and hence in horizontal spread of MDV to uninfected chickens.

Our data presented here suggest that US2, UL13, and gC are required in combination in order to facilitate horizontal transmission. The mechanism by which restoring and repairing the above genes leads to spread is unknown. One possibility could be a mere additive effect whereby each gene repaired increases replication and virulence such that spread ultimately becomes possible; however, it is difficult to imagine that the small increases in replication for each repair would suffice to facilitate horizontal transmission. A more likely mechanism is the functional involvement of US2, UL13, and gC in combination that allows the release of infectious virus or virus entry and/or uptake into cells of the naïve chicken. The functions that each of these genes perform are unknown at this time. Currently, pRB-1B-derived viruses are being generated that express gC and UL13, but not US2, to corroborate that the encoded protein is indeed dispensable for horizontal transmission of MDV in our system.


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ACKNOWLEDGMENTS
 
This work was supported in part by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2003-02234, and by PHS grant AI063048A to N.O.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. Phone: (607) 253-4067. Fax: (607) 253-3384. E-mail: kwj4{at}cornell.edu Back

{triangledown} Published ahead of print on 18 July 2007. Back


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Journal of Virology, October 2007, p. 10575-10587, Vol. 81, No. 19
0022-538X/07/$08.00+0     doi:10.1128/JVI.01065-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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