The requirement of glycoprotein C (gC) for interindividual spread is a conserved function of gC for avian herpesviruses

We have formerly shown that glycoprotein C (gC) of Gallid alphaherpesvirus 2, better known as Marek’s disease (MD) alphaherpesvirus (MDV), is required for interindividual spread in chickens. Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized gC was important for interindividual spread of other alphaherpesviruses. To test this hypothesis, we first generated a fluorescent protein tagged clone of Gallid alphaherpesvirus 3 MD vaccine strain 301B/1 to track virus replication in cell culture and chickens using fluorescent microscopy. Following validation of this system, we removed the open reading frame of 301B/1 gC from the genome and determined whether it was required for interindividual spread using experimental and natural infection studies. Interindividual spread of MD vaccine 301B/1 was abrogated by removal of 301B/1 gC. Rescuent virus in which 301B/1 gC was inserted back into the genome efficiently spread among chickens. To further study the conserved function of gC, we replaced 301B/1 gC with MDV gC and this virus also efficiently spread in chickens. These data suggest the essential function of alphaherpesvirus gC proteins is conserved and can be exploited during the generation of future vaccines against MD that affects the poultry industry worldwide.


We have formerly shown that glycoprotein C (gC) of Gallid alphaherpesvirus 2, better known as Marek's disease (MD) alphaherpesvirus (MDV), is required for interindividual spread in chickens.
Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized gC was important for interindividual spread of other alphaherpesviruses. To test this hypothesis, we first generated a fluorescent protein tagged clone of Gallid alphaherpesvirus 3 MD vaccine strain 301B/1 to track virus replication in cell culture and chickens using fluorescent microscopy. Following validation of this system, we removed the open reading frame of 301B/1 gC from the genome and determined whether it was required for interindividual spread using experimental and natural infection studies. Interindividual spread of MD vaccine 301B/1 was abrogated by removal of 301B/1 gC. Rescuent virus in which 301B/1 gC was inserted back into the genome efficiently spread among chickens. To further study the conserved function of gC, we replaced 301B/1 gC with MDV gC and this virus also efficiently spread in chickens. These data suggest the essential function of alphaherpesvirus gC proteins is conserved and can be exploited during the generation of future vaccines against MD that affects the poultry industry worldwide.
Herpesviruses have co-evolved with their respective hosts for millions of years. In most cases, each herpesvirus and host have reached a relatively stable relationship with many hosts infected with multiple herpesviruses, including humans that currently have nine associated herpesviruses 1 . Although there is a tremendous amount of information on herpesvirus-host interactions in cell culture, little is known about their relationships pertaining to interindividual spread and the viral and cellular genes that mediate this important aspect of the virus lifecycle. This is primarily due to the difficulty of studying the mechanistic nature of human herpesviruses in humans, as well as a lack of many natural animal models. For this, we turn to natural herpesvirus-host models to address questions on interindividual spread and dissemination in populations.
For much of the 1990s, there was a tremendous amount of attention paid to conserved herpesvirus glycoprotein C (gC) homologues due to their high expression levels and immunogenicity, where they were shown to perform multiple functions in vitro. Some of the functions identified include primary attachment of cell-free virus to heparin sulfate (HS)-and chondroitin-like glycosaminoglycans (GAGs) on the surface of cells 2,3 , and involvement in late steps of virus egress from cultured cells 2,4 . Although gC is not essential for most herpesviruses studied thus far, it significantly increases the efficiency of infection by providing an additional binding mechanism 5 and helps shield the virus from antibody neutralization 6 . In addition to viral attachment and egress, gC homologues are thought to have immune evasion functions mediated by binding to and inhibiting the action of complement component C3 [7][8][9][10][11][12] , as well as a role in chemokine-mediated leukocyte migration 13 . Similar to MDV, gC homologues for herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) appear to play a minor role in tissue culture model systems but are critical for HSV-1 and VZV replication in human skin cells using the SCID-hu mouse model 14 suggesting gC homologs may perform conserved functions during natural infection of the host. However, studies on the role of gC homologs are limited due to a lack of natural animal host model systems.  [15][16][17][18] . MDV causes Marek's disease in chickens, presenting with severe clinical symptoms including the development of solid lymphomas in the viscera and other organs; metabolic dysfunction; and neurological signs like paralysis and ataxia 19 . It is a major economic problem in the poultry industry due to its global distribution and transmissibility 20 . Natural infection of MDV begins through the respiratory route by inhalation of infectious virus where pulmonary B lymphocytes and macrophages or dendritic cells 21 are initially infected and transport the virus to lymphoid organs. Primary cytolytic infection ensues in activated T lymphocytes recruited to the sites of infection, which become the primary cell type infected, and latency is established in these cells. Depending on the line of chicken or MDV strain, oncogenic transformation of latently infected T cells results in lymphoma formation that is ultimately a dead-end for the virus. Important for dissemination in the population, migrating infected cells transport MDV to feather follicle (FF) epithelial (FFE) cells in the skin, where infectious cell-free virus is shed into the environment, and the virus life cycle can repeat in new hosts.
There are currently eight herpesviruses identified in avian species with all characterized within the subfamily Alphaherpesvirinae in the Herpesviridae family 1 . Of the eight avian herpesviruses, six belong to the Mardivirus genus of which MDV or Gallid alphaherpesvirus 2 (GaHV-2) is the prototypic virus within this genus. MD is controlled through vaccination with attenuated MDV strains and homologous non-oncogenic avian herpesviruses, including Gallid alphaherpesvirus 3 (GaHV-3) and turkey herpesvirus (HVT: Meleagrid alphaherpesvirus; MeHV-1). However, the current vaccines are efficient at reducing tumor formation and disease but not block interindividual spread of virulent MDV resulting in increasing virulence over the decades 22 . It is generally accepted that GaHV-3 and HVT have similar interindividual spread pathogeneses as MDV.
Here, we hypothesized that the absolute requirement of gC for MDV interindividual spread is conserved among other avian herpesviruses. To test this hypothesis, we used a recently generated infectious bacterial artificial chromosome (BAC) clone of the MD vaccine strain 301B/1 23 in experimental and natural infections of chickens to determine whether 301B/1 gC is required for 301B/1 transmission. Our results conclusively showed that 301B/1 gC is required for interindividual spread and that MDV gC could compensate for 301B1 gC in this process. These results suggest the importance of gC homologs in interindividual spread may be a conserved function and draws importance to studying this glycoprotein during interindividual spread of other herpesviruses.

Results
Generation of recombinant (r)301B/1 expressing pUL47mRFP (r301B47R). We and others have shown that fusing fluorescent proteins to the C-terminus of alphaherpesvirus pUL47 (VP13/14) allows the visualization of infected cells and does not affect replication in cell culture and in vivo for numerous herpesviruses [24][25][26][27][28] . Therefore, we inserted monomeric red fluorescent protein (mRFP) at the C-terminus of the pUL47 in a recently described BAC clone of 301B/1 23 to generate r301B47R (Fig. 1A). RFLP analysis confirmed the integrity of the BAC clones as the predicted banding pattern was observed described in the figure (Fig. 1B). In addition, DNA sequencing was used to confirm that each clone was correct at the nucleotide level (data not shown) using primers specific for each gene (Table 1).

Figure 1.
Generation of r301B/1clones. (A) Schematic representation of the 301B infectious clone 23 genome depicting the locations of the terminal repeat long (TRL) and short (TRS), internal repeat long (IRL) and short (IRS), and unique long (UL) and short (US) regions. The region of the UL spanning UL43 to UL50 is expanded to show the relevant genes within this region and modifications for each r301B/1 clone. (B) Predicted and actual RFLP analysis of r301B/1 clones. BAC DNA obtained for r301B, r301B47R-integrate clone and two resolved clones were digested with BamHI and electrophoresed through a 1.0% agarose gel. Integration of the mRFP + AphAI sequence resulted in an increase in the 9545 bp (blue colour rightwards arrow) fragment to 11,207 bp (yellow colour rightwards arrow). Resolution by removal of the AphAI sequence shifted the 11,207 bp fragment to 10,233 bp (red colour leftwards arrow). One resolved clone (#) was used after this point. (C) Predicted and actual RFLP analysis of r3ΔgC r301B clone. BAC DNA obtained for r301B47R, r3ΔgC-integrate clone and r3ΔgC-resolved clone were digested with BamHI and electrophoresed through a 1.0% agarose gel. Integration of the AphAI sequence into this locus removed a BamHI site combining the 10,223 and 14,854 bp fragments (blue colour rightwards arrow) to 24,671 bp (yellow colour rightwards arrow). Resolution of the AphAI sequence reduced the 23,933 bp fragment by 1028 bp to 23,643 bp (red colour leftwards arrow). (D) Predicted and actual RFLP analysis of r3ΔgC-R and r3-MDVgC clones derived from the r3ΔgC clone. BAC DNA obtained for r3ΔgC, r3ΔgC-R-integrate, r3ΔgC-R-resolved, r3-MDVgC-integrate, and r3-MDVgCresolved clones were digested with EcoRI and electrophoresed through a 1.0% agarose gel. Integration of 3 × Flag301BgC-AphAI or MDVgC-AphAI sequences into this locus resulted an increase in the 12,879 bp (blue colour rightwards arrow) fragment to 15,420 bp (yellow colour rightwards arrow) or 15,403 bp (yellow colour rightwards arrow), respectively. Removal of the AphAI sequence from r3ΔgC-R-Int reduced the 15,420 bp fragment by 1038 bp to 14,382 bp (red colour leftwards arrow) to generate r3ΔgC-R-Res. Removal of the    www.nature.com/scientificreports/ Additionally, while reconstituting r301B47R in DF-1-Cre (data not shown) and propagating in CEC cultures, mRFP was abundantly expressed that could be visualized using fluorescent microscopy ( Fig. 2B). Expression of pUL47mRFP was almost exclusively found in the nucleus, consistent with MDV, though the expression levels appeared to more abundant than observed for MDV 25,29 . Western blotting using anti-mRFP antibody showed mRFP expression was fused to the pUL47 since mRFP alone is ~ 26 kDa in size, while fused to pUL47 would create ~ 115 kDa protein (Fig. 2C). These results show that fusing mRFP to the C-terminus of pUL47 of 301B/1 resulted in no change in viral replication in tissue culture and allowed the direct visualization of 301B/1 replication in cells. This data is consistent with former studies fusing fluorescent proteins to pUL47 homologs 25-27,30 . v301B47R as a tool for tracking virus in chickens. Next, we tested the ability of v301B47R to replicate and interindividual spread in chickens. To do this, ten chickens were experimentally infected with 4000 PFU v301B47R and housed with ten naïve contact chickens for nine weeks to measure natural infection (interindividual spread). First, we were interested in whether we could directly identify infected birds based on fluorescence in plucked feathers as has been previously done in our laboratory for MDV 32 . Like our former results with fluorescently tagged MDV, feather follicles were easily identified from feathers ( Fig. 2D) in most experimentally infected chickens at 14 to 28 days post-infection (dpi) with a total of 90% of birds positive for v301B47R by 21 dpi (Fig. 2E). Staining of FFs showed feathers positive for pUL47mRFP were also positive for anti-GaHV-3 glycoprotein B (Y5.9), while negative for anti-HVT glycoprotein B (L78.2). These results show that fusing mRFP to 301B/1 pUL47 can be an effective tool to track 301B/1 in chickens.
v301B47R can spread from chicken to chicken. Over the course of 9 weeks, 50% of the naïve contact chickens housed with experimentally infected chickens became positive by the time the experiment was terminated ( Fig. 2E). There was a delay of about three weeks before naïve contact chickens began to show fluorescent feathers compared to experimentally infected chickens, which is consistent with the time it takes for MDV to interindividual spread. These results confirm that 301B/1 can interindividual spread from chicken to chicken.
Generation of r301B/1 lacking gC or expressing 3 × Flag301B gC or MDV gC. Now that we had a tool to track 301B/1 in cell culture and chickens, we wanted to test two hypotheses. First, we hypothesized that 301B/1 gC, like MDV gC, would be required for interindividual spread in chickens. Second, we hypothesized that, since both GaHV-3 and MDV are chicken herpesviruses with similar pathogeneses, MDV gC would compensate for 301B/1 replication and transmission. Therefore, we removed the complete UL44 (gC) open reading frame (ORF) from r301B47R to generate r3ΔgC (Fig. 1A). To generate a rescuent virus, 301B/1 gC was inserted back into the viral genome where it was originally removed but included a 3 × Flag tag at the N-terminus (r3-ΔgC-R) that should allow us to identify 301B/1 gC in downstream studies. In addition, we inserted MDV gC in its place to generate r3-MDVgC. RFLP analysis confirmed the integrity of the BAC clones as the predicted banding pattern was observed (Fig. 1C, D). In addition, DNA sequencing confirmed that each clone was correct at the nucleotide level (data not shown) using primers specific for each gene ( Table 1).
Replication of v301B/1 lacking gC (v3ΔgC) or expressing MDV gC (v3-MDVgC) in cell culture. Following reconstitution of r301B/1 clones with UL44 removed (v3ΔgC) and replaced with Flag-tagged 301B/1 gC (r3ΔgC-R) or MDV gC (r3-MDVgC), we tested replication in CEC cultures using plaque size assays (Fig. 3A). Removal of 301B/1 gC resulted in significantly large plaque sizes, which is consistent with what is observed for MDV 17,18 . Adding 3 × Flag301B gC restored smaller plaque sizes (v3ΔgC-R), while adding MDV gC also restored smaller plaque sizes that were significantly different to v3ΔgC. However, virus growth kinetics measuring viral DNA replication in qPCR assays showed no significant differences (Fig. 3B). Figure 3C shows western blotting of total protein and media from infected cells using anti-MDV gC and -Flag antibodies. The rescued 301B/1 gC could be detected using the anti-Flag antibody in both cellular protein extracts and infected cell media, suggesting 301B/1 gC is also secreted as has been previously shown for MDV gC 17,33 . We also confirmed MDV gC expression in both infected cells and was in the media of infected cells. Immunofluorescence assays (IFA) were used to examine expression in cells and showed that Flag-tagged 301B/1 gC (Fig. 3D) and MDV gC proteins (Fig. 3E) were detected as expected. These results show that adding the 3 × Flag epitope to the N-terminus 301B/1 gC did not affect 301B/1 replication in cell culture and allowed us to identify its expression in vitro. Also, 301B/1 expressing MDV gC did not affect replication based on plaque size assays and MDV gC was expressed in v3-MDVgC.

301B/1 gC is required for interindividual spread.
To test our hypotheses that GaHV-3 gC, like MDV gC, would be required for interindividual spread in chickens, we tested our newly derived v3ΔgC using our experimental and natural infection model for interindividual spread. To do this, 8-10 chickens were inoculated with 10,000 PFU of each virus and housed with 6-10 uninfected chickens over the course of 8 weeks.
Using qPCR assays to measure 301B/1 replication in the blood of experimentally infected chickens (Fig. 4A) and presence in FFs (Fig. 4B), no differences were seen between v301B47R, v3ΔgC, and v3ΔgC-R. However, when contact chickens were monitored for natural infection, no chickens housed with v3ΔgC-infected birds became infected compared to 88% and 60% of contact chickens were infected with v301B47R and v3ΔgC-R, respectively (Fig. 4B). Following termination, whole blood was collected from all contact chicken, serum was tested for anti-GaHV-3 antibodies using IFA and blood was used to measure 301B/1 viral DNA. It was confirmed all chickens negative for fluorescent FFs were also negative for anti-GaHV-3 antibodies and 301B/1 viral DNA in the blood (data not shown). IFA (Fig. 4C) and western blotting (Fig. 4E)  hypothesis that MDV gC would compensate for 301B/1 replication and transmission, we also tested v3-MDVgC in vivo. There was no difference in virus replication in the blood using qPCR assays (Fig. 4A) nor the ability to reach the FFs (Fig. 4B). Interestingly, v3-MDVgC was able to naturally infect chickens similar to v301B47R and v3ΔgC-R showing that MDV gC can compensate for 301B/1 in this essential function in vivo. IFA (Fig. 4D) and western blotting (Fig. 4E) were used to confirm MDV gC expression was maintained during replication in FFE cells. These results show that MDV gC can compensate for 301B/1 gC during 301B/1 MD vaccine strain interindividual spread in chickens.

Discussion
In this report, we tested the importance of the alphaherpesvirus conserved gC protein for interindividual spread of the MD vaccine strain 301B/1. In addition, we tested whether MDV gC could compensate for 301B/1 gC in transmission and whether N-terminal tagging of 301B/1 gC would affect its function during interindividual spread. We were able to confidently conclude that 301B/1 gC is required for interindividual spread of 301B/1 www.nature.com/scientificreports/ virus and the addition of a 3 × Flag epitope at the N-terminus did not affect its function during transmission. We were also able to conclude that MDV gC can compensate 301B/1 gC during interindividual spread of 301B/1 virus. This data, combined with our former work on MDV 15,16 , suggests the essential role for the alphaherpesvirus conserved gC during interindividual spread is a conserved function of avian herpesviruses. The exact role of MDV and 301B/1 gC during interindividual spread is not completely understood, but the absolute requirement during natural infection suggests it may be involved in virus-cell binding to cells. Homologs of gC perform multiple functions in vitro that include primary attachment of cell-free virus to proteoglycans on the surface of cells 2,3,34,35 but is not required for specific interactions on cells where glycoprotein (gD) normally performs this function 36,37 . For HSV-1, gD binds to the herpesvirus entry mediator (HVEM), nectin-1, nectin-2, or modified heparin sulfate on the surface of cells providing a mechanism for cell tropic binding 38 and it is believed gD of other members of the Alphaherpesvirinae perform similar functions. However, formerly we have shown that gD is not required for MDV interindividual spread 15 . The absolute requirement for MDV, and now 301B/1, gC in this process suggests it plays a more direct role in binding to cells during MDV and 301B/1 natural infection. Based on the ability of 301B/1 to naturally infect chickens when expressing 301B/1 or MDV gC and the close sequence homology (72.655% protein identity) between the two proteins (Fig. 1E), both proteins may target the same cellular protein and cell type to initiate infection. We are currently performing studies to elucidate potential binding partners for gC.
Currently, most MD vaccines do not transmit efficiently in chickens and thus cannot compete with virulent virus that does spread efficiently. Read et al. 39 showed that current MD vaccines can enhance transmission of virulent MDV in the field, possibly because they are unable to block infection of chickens and shedding of virus. The fact that 301B/1 is as effective as traditional vaccine strains 23 and is able to efficiently transmit in chickens suggests this vaccine strain may better protect unvaccinated or "missed" chickens in a flock and potentially compete with virulent MDV for replication in the skin. On top of that, swapping MDV gC for 301B/1 will most likely provide better immunogenic responses to MDV as gC is a major antigenic target against MDV 40 and could increase its protective index. www.nature.com/scientificreports/ Another important result in this report was that both 301B/1 and MDV gC proteins were secreted into the infected culture cell media suggesting alternative splicing of 301B/1 and MDV gC occurs in 301B/1 as has been shown for MDV gC during MDV cell culture propagation 17 . Further analysis on the splicing of the gC mRNA during 301B/1 replication is warranted; however, this data suggests the splicing of gC transcripts is also conserved in 301B/1 and the mechanism of gC transcript splicing, such as ICP27 and pUL47 32,41 , may also be conserved among the avian herpesviruses.
In summary, our results support our hypothesis that the absolute requirement of gC during interindividual spread is conserved among alphaherpesviruses. This report extends our work on MDV gC requirement to the MD vaccine strain 301B/1, and we can conclude at least for some avian herpesviruses with similar pathogeneses, that the functional importance of gC during interindividual spread is conserved. Further studies are warranted to determine whether gC homologs of other alphaherpesvirus are required for natural infection, although there are limited natural animal models to perform such studies making the avian herpesvirus models important for understanding conserved herpesvirus genes during natural infections.
The chicken DF-1-Cre fibroblast cell line 43

Generation of two-step red recombination shuttle vectors.
To produce pEP-301BgC-in, 301B/1 UL44 was Gibson assembly cloned from r301B/1 BAC DNA into pcDNA3.1 using primers shown in Table 2. Briefly, 301B/1 UL44 was amplified by PCR using a set of primers encompassing the complete UL44 gene, gel purified, and cloned into the pcDNA3.1 TOPO vector (Life Technologies) using Gibson Assembly reaction mix (NEB) according to the manufacturer's instructions to generate pc301BgC. Next, a 3 × Flag epitope was cloned into pc301BgC from a previously described r3 × Flag54 BAC clone 32 using Gibson assembly to generate pc3 × Flag301BgC. Next, the aphAI-I-SceI cassette was amplified from pEP-KanS2 using primers shown in Table 2 and inserted into pc3 × Flag301BgC using Gibson Assembly cloning to generate pEP-301BgC-in. All clones at each step were confirmed by PCR and DNA sequencing. For insertion of MDV gC (RB1B strain) into r301B/1, a previously described pEP-MDVgC-in shuttle vector was used 17 .
Generation of r301B/1 clones. To create 301B/1 expressing fluorescent-tagged pUL47, the coding sequence of the monomeric red fluorescent protein (mRFP) gene was inserted in frame at the C-terminus of the 301B/1 UL47 ORF by two-step Red-mediated mutagenesis 44 in an infectious BAC clone of 301B/1. Briefly, the mRFP-I-SceI-aphAI cassette was amplified from pEP-mRFP-in 45 using primers shown in Table 3 and used for mutagenesis in GS1783 Escherichia coli cells. Multiple integrates and resolved clones were screened by RFLP analysis, analytic PCR, and DNA sequencing using primers shown in Table 1. Table 2. Primers used for cloning and generation of shuttle vectors using Gibson assembly cloning. a Construct generated with the set of primers. b Directionality of the primer and product produced for Gibson assembly cloning.  Vector For  ACA TAT TAC TTT CGT CCG TCG GTA AGC CTA TCC CTA ACC CTC TCC   Vector Rev  GAC GCG TGC ATG GGG AAA ATT CCG AGC TCG GTA CCA AGC TTA ACTAG   Insert For  AGC TTG GTA CCG AGC TCG GAA TTT TCC CCA TGC ACG CGT CAC G   Insert Rev  GGG TTA GGG ATA GGC TTA CCG ACG GAC GAA AGT AAT ATG TAT TTT TTC CCGG   pc3 × Flag301B gC   Vector For  ACA AGG ATG ACG ACG ATA AGA TTA ACC CCG ATC TAG CTA CAC CC   Vector Rev  CCG TCA TGA TCC TTG TAA TCG CTA GCG CTT AGG ACG CG   Insert For  GCC GCG TCC TAA GCG CTA GCG ATT ACA AGG ATC ATG ACG GAG ATT ACA AGG   Insert Rev  GTA GCT AGA TCG GGG TTA ATC TTA TCG TCG TCA TCC TTG TAA TCG ATG T  www.nature.com/scientificreports/ To create r3ΔgC, the coding sequence of 301B/1 UL44 (gC) was deleted from r301B47R. Briefly, the I-SceI-aphAI cassette from pEP-KanS2 was amplified by PCR with Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix using primers shown in Table 3 and used for mutagenesis in GS1783 E. coli cells. Following removal of UL44 in the r301B47R clone, 301B/1 gC with a 3 × Flag epitope inserted at the N-terminus after the predicted signal sequence, or MDV gC were inserted into r3ΔgC using two-step Red recombination. Briefly, 3 × Flag301B/1 gC or MDV gC were PCR amplified from pEP-301BgC-in or pEP-MDVgC-in, respectively, using primers shown in Table 3 and used for mutagenesis as described above. RFLP analysis, analytical PCR, and DNA sequencing confirmed all clones were correct. Primers used for MDV gC have been previously published 17,46,47 , while primers for sequencing 301B/1 gC are listed in Table 1. r301B/1 s were reconstituted by transfecting DF-1-Cre cells with purified BAC DNA plus Lipofectamine 2000 (Invitrogen) using the manufacturers' instructions as previously described 29 . Transfected DF-1-Cre cells were mixed with fresh primary CEC cultures until plaques formed, then further propagated in CEC cultures until virus stocks could be stored. All viruses were used at ≤ 5 passages for in vitro and in vivo studies. Viral growth kinetics in cell culture. To measure viral growth kinetics of viruses in cell culture, qPCR assays were used to measure the relative level of replication as previously described 23 . Briefly, CEC cultures were prepared in 6-well tissue culture plates and the next day inoculated with 100 PFU/well. Total DNA was collected from the inoculum and at 48, 72, 96, and 120 h following infection using the QIAamp DNA Mini Kit (Qiagen, Germantown, MD). Quantification of 301B/1 genomic copies in CEC cultures was performed using primers and probe previously described 23 and were used in duplex PCR reactions with previously described primers and probes against chicken iNOS 51 . All qPCR assays were performed in an Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) and the results were analyzed using the QuantStudio Design & Analysis Software v1.4.2 supplied by the manufacturer. The fold-increase in viral DNA copies over inoculum was used to determine differences in replication.

Western blot analysis.
Western blot analyses were performed as previously described 48 . Total protein was collected from infected CEC cultures or scraped from FFE previously described 18 . In some experiments, infected  AGA AGA TGC GAA GGA GGC GAT CTT CAA AAA AAC GGA CCGG ATG GCC TCC TCC GAG GAC G   Reverse  TCA CCA CGA TCT GCA CGC CGC TCC GTG CGC TTT TTT TTTA CAA GGC GCC GGT GGA GTG   ΔgC   Forward  ATA TAC GCT CTC GGA GAC GCG GCT CGC ACG CCA GCT GAA ATA TTT TCC CCTAG TTT GCG GTG ACA TTGAT TAG GGA TAA CAG  GGT AAT CGA TTT   Reverse  TAC AAG AGC TCG GGG CAT ATA ATG AGC CAG ATC AAT GTC ACC GCA AAC TAG GGG AAA ATA TTT CAG CTGG GCC AGT GTT ACA ACC  AAT  Animal experiments. Pure Columbian (PC) chickens were obtained from the UIUC Poultry Farm (Urbana, IL) and were from MD-vaccinated parents; therefore, considered to be maternal antibody positive. To test replication of v301B47R, 7-day old chicks (n = 10) were experimentally infected by intra-abdominal inoculation of 4000 PFU for v301B47R and housed with another ten chickens that were left uninfected to act as contacts to determine whether v301B47R can naturally infect naïve chickens by interindividual spread. To test the ability of v301B47R, v3ΔgC, v3ΔgC-R, v3-MDVgC to replicate and interindividual spread in chickens, 3-day old PC chicks (n = 8-10/group) were inoculated with 10,000 PFU with each respective virus and housed in separate rooms. To test natural infection through interindividual spread, 6-8 age-match, naïve contact chickens were housed experimentally infected chickens for eight weeks. Water and food were provided ad libitum for all animal experiments.
DNA extraction from blood cells and qPCR assays. Whole blood was obtained by wing-vein puncture and DNA was extracted using the E.Z. 96 blood DNA kit from Omega Bio-tek, Inc. (Norcross, GA) as previously described 15 . Quantification of 301B/1 genomic copies in the blood using qPCR was performed using primers and probe previously described 23 and were used in duplex PCR reactions with previously described primers and probes against chicken iNOS 51 . All qPCR assays were performed in an Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) and the results were analyzed using the QuantStudio Design & Analysis Software v1.4.2 supplied by the manufacturer. The final viral loads were obtained after normalizing with chicken iNOS used as an internal control gene.

Monitoring v301B47R and its derivatives in feather follicles (FFs).
To track the time at which each r301B47R or its derivatives reached the FFs, two flight feathers were plucked from the right and left wings (4 total) of inoculated birds weekly and pUL47mRFP expression was examined using a Leica M205 FCA fluorescent stereomicroscope with a Leica DFC7000T digital color microscope camera (Leica Microsystems, Inc., Buffalo Grove, IL).

IFA of feather follicles (FFs).
Whole feathers were plucked from chickens infected with different r301B/1s and the FFs were fixed using PFA buffer, washed twice with PBS, and then blocked in 10% neonatal calf serum (Sigma-Aldrich). Fixed FFs were stained with primary mouse anti-Flag M2 (Sigma-Aldrich) or anti-MDV gC A6 48 antibodies with anti-mouse Ig Alexa Fluor 488 (Molecular Probes, Eugene, OR) used as secondary antibody. The Leica M205 FCA fluorescent stereomicroscope with a Leica DFC7000T digital color microscope camera (Leica Microsystems, Inc., Buffalo Grove, IL) was used to analyze stained FFEs. All images were compiled using Adobe Photoshop version 21.0.1.

Statistical analyses.
Statistical analyses were performed using IBM SPSS Statistics Version 27 software (SPSS Inc., USA). Plaque size assays were analyzed with Student's t tests and one-way analysis of variance (ANOVA), virus was included as fixed effect and the plaque size was used as dependent variable. The normalized data of viral replication (qPCR) were analyzed using two-way ANOVA followed by LSD and Tukey's post hoc tests; virus (V) and time (T) and all possible interactions (V × T) were used as fixed effects, and the genomic copies as dependent variable. Fisher's exact test was used for infection and transmission experiments. Statistical significance was declared at p < 0.05 and the mean tests experiments associated with significant interaction (p < 0.05) were separated with Tukey's test.