Distinct polymorphisms in a single herpesvirus gene are capable of enhancing virulence and mediating vaccinal resistance.

Modified-live herpesvirus vaccines are widely used in humans and animals, but field strains can emerge that have a higher virulence and break vaccinal protection. Since the introduction of the first vaccine in the 1970s, Marek's disease virus overcame the vaccine barrier by the acquisition of numerous genomic mutations. However, the evolutionary adaptations in the herpesvirus genome responsible for the vaccine breaks have remained elusive. Here, we demonstrate that point mutations in the multifunctional meq gene acquired during evolution can significantly alter virulence. Defined mutations found in highly virulent strains also allowed the virus to overcome innate cellular responses and vaccinal protection. Concomitantly, the adaptations in meq enhanced virus shedding into the environment, likely providing a selective advantage for the virus. Our study provides the first experimental evidence that few point mutations in a single herpesviral gene result in drastically increased virulence, enhanced shedding, and escape from vaccinal protection.

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

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

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

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

A deletion in the glycoprotein L (gL) gene of U.S. Marek's disease virus (MDV) field strains is insufficient to confer increased pathogenicity to the bacterial artificial chromosome (BAC)-based strain, RB-1B.

Marek's disease (MD) is a highly transmissible, herpesvirus-associated malignancy of chickens and turkeys caused by Marek's disease virus (MDV). MD is currently controlled through the use of nonsterilizing vaccines composed of antigenically related, apathogenic herpesviruses Mardivirus 2 (MDV-2), Meleagrid herpesvirus 1 (herpesvirus of turkeys, HVT), or attenuated MDV-1 strain CVI988 (Rispens). Since the mid-1960s, field strains of MDV have increased in virulence, due, in part, to the widespread use of vaccines since the early 1970s. One mutation that we have identified common to very virulent field strains (vv and vv+MDVs) since the 1990s has been a mutation in the UL1 gene, encoding glycoprotein L (gL). This mutation, a 12-nucleotide (nt) deletion in the signal peptide of gL, has been associated with increased virulence and decreased vaccine protection in the context of challenge with a vv+MDV, strain TK. To determine whether this mutation alone was sufficient to confer increased virulence, we introduced this mutation into the transmission-competent pRB-1B bacterial artificial chromosome (BAC) using two-step, Red-mediated recombination. The resulting mutant, pRB-1BgLdelta, was tested for changes in replication in cell culture using multistep growth curves, plaque size analysis, viral burst analysis, and the ability to compete with the parental virus when co-transfected at different ratios and sequentially passaged. In addition, we examined this mutant for changes in pathogenicity in inoculated and contact-exposed unvaccinated and vaccinated chickens. Our data show minor differences in plaque sizes in cell culture, but no discernible changes in the infection of specific-pathogen-free (SPF) leghorn chickens. We therefore conclude that although this mutation is indeed common to MDV field strains isolated in the eastern United States, it is insufficient to confer increased virulence or loss of vaccine protection previously observed for a vv+MDV strain having this mutation.

A Special Issue on Marek's Disease Virus-The Editors' View.

Marek's disease virus (MDV), an Alphaherpesvirus belonging to the genus Mardivirus, causes T cell lymphomas in chickens and remains one of the greatest threats to poultry production worldwide [...].

Innate immune pathway modulator screen identifies STING pathway activation as a strategy to inhibit multiple families of arbo and respiratory viruses.

RNA viruses continue to remain a threat for potential pandemics due to their rapid evolution. Potentiating host antiviral pathways to prevent or limit viral infections is a promising strategy. Thus, by testing a library of innate immune agonists targeting pathogen recognition receptors, we observe that Toll-like receptor 3 (TLR3), stimulator of interferon genes (STING), TLR8, and Dectin-1 ligands inhibit arboviruses, Chikungunya virus (CHIKV), West Nile virus, and Zika virus to varying degrees. STING agonists (cAIMP, diABZI, and 2',3'-cGAMP) and Dectin-1 agonist scleroglucan demonstrate the most potent, broad-spectrum antiviral function. Furthermore, STING agonists inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enterovirus-D68 (EV-D68) infection in cardiomyocytes. Transcriptome analysis reveals that cAIMP treatment rescue cells from CHIKV-induced dysregulation of cell repair, immune, and metabolic pathways. In addition, cAIMP provides protection against CHIKV in a chronic CHIKV-arthritis mouse model. Our study describes innate immune signaling circuits crucial for RNA virus replication and identifies broad-spectrum antivirals effective against multiple families of pandemic potential RNA viruses.

A virus-encoded telomerase RNA promotes malignant T cell lymphomagenesis.

Telomerase is a ribonucleoprotein complex consisting of two essential core components: a reverse transcriptase and an RNA subunit (telomerase RNA [TR]). Dysregulation of telomerase has been associated with cell immortalization and oncogenesis. Marek's disease herpesvirus (MDV) induces a malignant T cell lymphoma in chickens and harbors in its genome two identical copies of a viral TR (vTR) with 88% sequence identity to chicken TR. MDV mutants lacking both copies of vTR were significantly impaired in their ability to induce T cell lymphomas, although lytic replication in vivo was unaffected. Tumor incidences were reduced by >60% in chickens infected with vTR- viruses compared with animals inoculated with MDV harboring at least one intact copy of vTR. Lymphomas in animals infected with the vTR- viruses were also significantly smaller in size and less disseminated. Constitutive expression of vTR in the chicken fibroblast cell line DF-1 resulted in a phenotype consistent with transformation as indicated by morphological alteration, enhanced anchorage-independent cell growth, cell growth beyond saturation density, and increased expression levels of integrin alpha v. We concluded that vTR plays a critical role in MDV-induced T cell lymphomagenesis. Furthermore, our results provide the first description of tumor-promoting effects of TR in a natural virus-host infection model.

Selection of a recombinant Marek's disease virus in vivo through expression of the Marek's EcoRI-Q (Meq)-encoded oncoprotein: characterization of an rMd5-based mutant expressing the Meq of strain RB-1B.

Marek's disease (MD) is a highly contagious viral disease of chickens (Gallus gallus domesticus) caused by MD virus (MDV), characterized by paralysis, neurologic signs, and the rapid onset of T-cell lymphomas. MDV-induced T-cell transformation requires a basic leucine zipper protein called Marek's EcoRI-Q-encoded protein (Meq). We have identified mutations in the coding sequence of Meq that correlated with virus pathotype (virulent, very virulent, and very virulent plus). The aim of this study was to determine whether recombinant viruses could be isolated based on Meq expression through in vivo selection. Chicken embryo fibroblasts (CEFs) were cotransfected with an rMd5 strain-based Meq deletion virus (rMd5deltaMeq) and meq loci from strains representing different pathotypes of MDV. Transfected CEFs were inoculated into chickens in two independent studies. We were able to isolate a single recombinant virus, rMDV-1137, in a contact-exposed chicken. rMDV-1137 had recombined two copies of the meq gene of RB-1B and was found to have pathogenicity similar to both RB-1B and rMd5 parental strains. We found the RB-1B- and rMd5-induced lymphomas showed differences in composition and that rMDV-1137-induced lymphomas were intermediate in their composition. We were able to establish cell lines from both RB-1B- (MDCC-UD35, -UD37) and rMDV-1137 (MDCC-UD36, -UD38)-induced, but not rMd5-induced, lymphomas. To date, no rMd5- or parent Md5-transformed T-cell lines have been reported. Our results suggest that 1) a recombinant MDV can be selected on the basis of oncogenicity; 2) changes in Meq sequence seem to affect tumor composition and the ability to establish cell lines; and 3) in addition to meq, other genomic loci affect MDV pathogenicity and oncogenicity.

Effect of Insertion and Deletion in the Meq Protein Encoded by Highly Oncogenic Marek's Disease Virus on Transactivation Activity and Virulence.

Marek's disease virus (MDV) causes malignant lymphoma in chickens (Marek's disease, MD). Although MD is currently controlled by vaccination, MDV strains have continuously increased in virulence over the recent decades. Polymorphisms in Meq, an MDV-encoded oncoprotein that serves as a transcription factor, have been associated with the enhanced virulence of the virus. In addition, insertions and deletions in Meq have been observed in MDV strains of higher virulence, but their contribution to said virulence remains elusive. In this study, we investigated the contribution of an insertion (L-Meq) and a deletion in the Meq gene (S-Meq) to its functions and MDV pathogenicity. Reporter assays revealed that both insertion and deletion enhanced the transactivation potential of Meq. Additionally, we generated RB-1B-based recombinant MDVs (rMDVs) encoding each Meq isoform and analyzed their pathogenic potential. rMDV encoding L-Meq indueced the highest mortality and tumor incidence in infected animals, whereas the rMDV encoding S-Meq exhibited the lowest pathogenicity. Thus, insertion enhanced the transactivation activity of Meq and MDV pathogenicity, whereas deletion reduced pathogenicity despite having increased transactivation activity. These data suggest that other functions of Meq affect MDV virulence. These data improve our understanding of the mechanisms underlying the evolution of MDV virulence.

A Rat Model of Prenatal Zika Virus Infection and Associated Long-Term Outcomes.

Zika virus (ZIKV) is a mosquito-borne flavivirus that became widely recognized due to the epidemic in Brazil in 2015. Since then, there has been nearly a 20-fold increase in the incidence of microcephaly and birth defects seen among women giving birth in Brazil, leading the Centers for Disease Control and Prevention (CDC) to officially declare a causal link between prenatal ZIKV infection and the serious brain abnormalities seen in affected infants. Here, we used a unique rat model of prenatal ZIKV infection to study three possible long-term outcomes of congenital ZIKV infection: (1) behavior, (2) cell proliferation, survival, and differentiation in the brain, and (3) immune responses later in life. Adult offspring that were prenatally infected with ZIKV exhibited motor deficits in a sex-specific manner, and failed to mount a normal interferon response to a viral immune challenge later in life. Despite undetectable levels of ZIKV in the brain and serum in these offspring at P2, P24, or P60, these results suggest that prenatal exposure to ZIKV results in lasting consequences that could significantly impact the health of the offspring. To help individuals already exposed to ZIKV, as well as be prepared for future outbreaks, we need to understand the full spectrum of neurological and immunological consequences that could arise following prenatal ZIKV infection.

An Examination of the Long-Term Neurodevelopmental Impact of Prenatal Zika Virus Infection in a Rat Model Using a High Resolution, Longitudinal MRI Approach.

Since Zika virus (ZIKV) first emerged as a public health concern in 2015, our ability to identify and track the long-term neurological sequelae of prenatal Zika virus (ZIKV) infection in humans has been limited. Our lab has developed a rat model of maternal ZIKV infection with associated vertical transmission to the fetus that results in significant brain malformations in the neonatal offspring. Here, we use this model in conjunction with longitudinal magnetic resonance imaging (MRI) to expand our understanding of the long-term neurological consequences of prenatal ZIKV infection in order to identify characteristic neurodevelopmental changes and track them across time. We exploited both manual and automated atlas-based segmentation of MR images in order to identify long-term structural changes within the developing rat brain following inoculation. The paradigm involved scanning three cohorts of male and female rats that were prenatally inoculated with 107 PFU ZIKV, 107 UV-inactivated ZIKV (iZIKV), or diluent medium (mock), at 4 different postnatal day (P) age points: P2, P16, P24, and P60. Analysis of tracked brain structures revealed significantly altered development in both the ZIKV and iZIKV rats. Moreover, we demonstrate that prenatal ZIKV infection alters the growth of brain regions throughout the neonatal and juvenile ages. Our findings also suggest that maternal immune activation caused by inactive viral proteins may play a role in altered brain growth throughout development. For the very first time, we introduce manual and automated atlas-based segmentation of neonatal and juvenile rat brains longitudinally. Experimental results demonstrate the effectiveness of our novel approach for detecting significant changes in neurodevelopment in models of early-life infections.

An Engineered Receptor-Binding Domain Improves the Immunogenicity of Multivalent SARS-CoV-2 Vaccines.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication.

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

A comparison of exosome purification methods using serum of Marek's disease virus (MDV)-vaccinated and -tumor-bearing chickens.

Marek's disease (MD) is an alphaherpesvirus (Marek's disease virus, MDV)-induced pathology of chickens associated with paralysis, immunosuppression, neurological signs, and T-cell lymphomas. MD is controlled in poultry production via live attenuated vaccines. The purpose of the current study was to compare methods for precipitating exosomes from vaccinated and protected chicken sera (VEX) and tumor-bearing chicken sera (TEX) for biomarker analysis of vaccine-induced protection and MD lymphomas respectively. A standard polyethylene glycol (PEG, 8%) method was compared to a commercial reagent (total exosome isolation reagent, TEI) for exosome yield and RNA content. Although exosomes purified by PEG or TEI were comparable in size and morphology, TEI-reagent yielded 3-4-fold greater concentration. Relative expression of 8 out of 10 G. gallus- and MDV1-encoded miRNAs examined displayed significant difference depending upon the precipitation method used. Standard PEG yields comparable, albeit lower amounts of exosomes than the TEI-reagent and a distinctive miRNA composition.

An engineered receptor-binding domain improves the immunogenicity of multivalent SARS-CoV-2 vaccines.

The SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD expresses inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) expresses markedly more efficiently, and generates a more potent neutralizing responses as a DNA vaccine antigen, than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers such as an H. pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.

A deletion within glycoprotein L of Marek's disease virus (MDV) field isolates correlates with a decrease in bivalent MDV vaccine efficacy in contact-exposed chickens.

We examined the functional role of a naturally occurring deletion within the glycoprotein L (gL) gene of Marek's disease virus (MDV) field isolates. We previously showed that this mutation incrementally increased the virulence of an MDV in contact-exposed SPF leghorn chickens, when chickens shedding this virus were co-infected with herpesvirus of turkeys (HVT). In our present study, we examined this mutation using two stocks of the very virulent plus (vv+)MDV strain TK, one of which harbored this deletion (TK1a) while the other did not (TK2a). We report that TK1a replicating in vaccinated chickens overcame bivalent (HVT/SB1) vaccine protection in contact-exposed chickens. Treatment groups exposed to vaccinated chickens inoculated with a 1:1 mix of TK1a and TK2a showed decreased bivalent vaccine efficacy, and this decrease correlated with the prevalence of the gL deletion indicative of TK1a. These results were also found using quadruplicate treatment groups and bivalently vaccinated chickens obtained from a commercial hatchery. As this deletion was found in 25 out of 25 recent field isolates from Delaware, Maryland, North Carolina, Pennsylvania, and Virginia, we concluded that there is a strong selection for this mutation, which appears to have evolved in HVT or bivalently vaccinated chickens. This is the first report of a mutation in a vv+MDV field strain for which a putative biological phenotype has been discerned. Moreover, this mutation in gL has apparently been selected in MDV field isolates through Marek's disease vaccination.

Latency of Marek's disease virus (MDV) in a reticuloendotheliosis virus-transformed T-cell line. I: uptake and structure of the latent MDV genome.

Marek's disease virus (MDV) is an acute transforming alphaherpesvirus of chickens that causes Marek's disease. During the infection of chickens, MDV establishes latency in CD4+ (T-helper) cells, which are also the target of transformation. The study of MDV latency has been limited to the use of MDV tumor-derived cell lines or blood cells isolated from chickens during presumed periods of latent infection. In 1992 Pratt et al. described the uptake of the MDV genome by a reticuloendotheliosis-transformed T-cell line (RECC-CU91). They reported that MDV established latency in CU91 cells, but that MDV genome expression was very limited. In this report we have examined the uptake of oncogenic, recombinant, and vaccine strain MDVs. We report that the entire MDV genome is taken up by CU91 cells, is hypomethylated, and readily reactivates from this latent state in a manner similar to MDV-transformed cell lines. Notably, virus could not be recovered from cell lines harboring vaccine virus CVI988 or the JM102 strain of MDV. Overall these cell lines present a useful model for the further study of MDV latency, particularly for those viruses having mutations that may affect replication or fitness of the virus in vivo. In addition, these cell lines offer an attractive means to study the latency of vaccine viruses, which establish relatively low levels of latent infection in vivo.

Latency of Marek's disease virus (MDV) in a reticuloendotheliosis virus-transformed T-cell line. II: expression of the latent MDV genome.

Marek's disease virus (MDV) is an alphaherpesvirus of chickens that causes the paralysis and rapid lymphoma formation known as Marek's disease. MDV establishes latent infection in activated CD4+ T-cells, and these cells are also the target for transformation. MDV latency has been studied using MDV lymphoma-derived cell lines and T-cells isolated from infected chickens. Each of these models has limitations because MDV-transformed cell lines require the use of oncogenic viruses; conversely, pools of latently infected cells are in relatively low abundance and invariably contain cells undergoing reactivation to lytic infection. In this study we have examined the spontaneous and induced expression of the MDV genome, the effect of genome uptake on cellular proliferation and apoptosis resistance, and differences in cellular surface antigen expression associated with MDV genome uptake in a reticuloendotheliosis virus (REV)-transformed T-cell model. We report that the MDV genome is highly transcribed during this latent infection, and that the expression of Marek's EcoRI-Q-encoded protein (Meq) transcripts is similar to that of MDV-transformed cells, but is somewhat lower than MDV-transformed cells at the protein level. Uptake of the MDV genome was associated with an increased growth rate and resistance to serum starvation-induced apoptosis. Treatment of cells with bromodeoxyuridine induced the expression of MDV lyric antigens in a manner similar to MDV-transformed cells. Uptake of the MDV genome, however, was not consistently associated with alteration ofT-cell surface antigen expression. Overall, our data show that the REV-transformed cell line model for MDV latency mimics many important aspects of latency also observed in MDV-transformed cells and provides an additional tool for examining MDV latent infection.

Comparison of the Transcriptomes and Proteomes of Serum Exosomes from Marek's Disease Virus-Vaccinated and Protected and Lymphoma-Bearing Chickens.

Marek's disease virus (MDV) is the causative agent of Marek's disease (MD), a complex pathology of chickens characterized by paralysis, immunosuppression, and T-cell lymphomagenesis. MD is controlled in poultry production via vaccines administered in ovo or at hatch, and these confer protection against lymphoma formation, but not superinfection by MDV field strains. Despite vaccine-induced humoral and cell-mediated immune responses, mechanisms eliciting systemic protection remain unclear. Here we report the contents of serum exosomes to assess their possible roles as indicators of systemic immunity, and alternatively, tumor formation. We examined the RNA and protein content of serum exosomes from CVI988 (Rispens)-vaccinated and protected chickens (VEX), and unvaccinated tumor-bearing chickens (TEX), via deep-sequencing and mass spectrometry, respectively. Bioinformatic analyses of microRNAs (miRNAs) and predicted miRNA targets indicated a greater abundance of tumor suppressor miRNAs in VEX compared to TEX. Conversely, oncomiRs originating from cellular (miRs 106a-363) and MDV miRNA clusters were more abundant in TEX compared to VEX. Most notably, mRNAs mapping to the entire MDV genome were identified in VEX, while mRNAs mapping to the repeats flanking the unique long (IRL/TRL) were identified in TEX. These data suggest that long-term systemic vaccine-induced immune responses may be mediated at the level of VEX which transfer viral mRNAs to antigen presenting cells systemically. Proteomic analyses of these exosomes suggested potential biomarkers for VEX and TEX. These data provide important putative insight into MDV-mediated immune suppression and vaccine responses, as well as potential serum biomarkers for MD protection and susceptibility.

Transcriptional Analyses of Innate and Acquired Immune Patterning Elicited by Marek's Disease Virus Vaccine Strains: Turkey Herpesvirus (HVT), Marek's Disease Virus 2 (strain SB1), and Bivalent Vaccines (HVT/SB1 and HVT-LT/SB1).

Marek's disease (MD) is a complex pathology of chickens caused by MD virus (MDV) 1 and is observed as paralysis, immune suppression, neurologic signs, and the rapid formation of T-cell lymphomas. The incidence of MD in commercial broilers is largely controlled via vaccination, either in ovo or at hatch with live attenuated vaccines, i.e., turkey herpesvirus (HVT) or a bivalent combination of HVT with the MDV 2 strain (SB1). To further extend the protection conferred by bivalent HVT/SB-1, recombinant HVTs encoding transgenes of other avian viruses have similarly been used for in ovo administration. Despite decades of use, the specific mechanisms associated with vaccine-induced protection remain obscure. Additionally, the mechanistic basis for vaccine synergism conferred by bivalent HVT/SB-1, compared with HVT or SB-1 administered alone, is largely unknown. In the present study, we report on temporal changes in innate and acquired immune-patterning gene expression by using ex vivo splenocyte infection and in ovo vaccination models. We report that in the ex vivo splenocyte infection model, by 72 hr postinfection, vaccines induced IFN and IFN-stimulated gene expression, with lesser proinflammatory cytokine induction. For several genes (TLR3, IFN-γ, OASL, Mx1, NOS2A, and IL-1ß), the effects on gene expression were additive for HVT, SB1, and HVT/SB1 infection. We observed similar patterns of induction in in ovo-vaccinated commercial broiler embryos and chicks with HVT/SB-1 or recombinant HVT-based bivalent combination (HVT-LT/SB-1). Furthermore, HVT/SB-1 or HVT-LT/SB-1 in ovo vaccination appeared to hasten immune maturation, with expression patterns suggesting accelerated migration of T and natural killer cells into the spleen. Finally, HVT/SB-1 vaccination resulted in a coordinated induction of IL-12p40 and downregulation of suppressors of cytokine signaling 1 and 3, indicative of classical macrophage 1 and T-helper 1 patterning.

Análisis transcripcionales de patrones inmunes innatos y adquiridos inducidos por cepas vacunales del virus de la enfermedad de Marek: virus herpes del pavo (HVT), virus de Marek 2 (cepa SB1) y vacunas bivalentes (HVT/SB1 y HVT-LT/SB1). La enfermedad de Marek (MD) es una patología compleja de los pollos causada por el virus de Marek (MDV) 1 y se observa como parálisis, depresión inmune, signos neurológicos y la formación rápida de linfomas de células T. La incidencia de la enfermedad de Marek en pollos de engorde comerciales se controla en gran medida a través de la vacunación, ya sea in ovo o al momento de la eclosión con vacunas vivas atenuadas, por ejemplo, herpesvirus de pavo (HVT) o una combinación bivalente de HVT con la cepa SB1. Para ampliar aún más la protección conferida por la vacuna bivalente HVT/SB-1, los HVT recombinantes que codifican transgenes de otros virus aviares se han utilizado de forma similar para la administración in ovo. A pesar de décadas de uso, los mecanismos específicos asociados con la protección inducida por la vacuna siguen sin ser esclarecidos completamente. Además, el mecanismo para la sinergia de la vacuna conferida por la vacuna bivalente HVT/SB-1, en comparación con la administración de la cepa HVT o de la cepa SB-1 por sí solas, es en gran medida desconocida. En el presente estudio, se informa sobre los cambios temporales en la expresión genética de patrones inmunes innatos y adquiridos mediante la infección de esplenocitos ex vivo y en modelos de vacunación in ovo. Se reporta que en el modelo de infección de esplenocitos ex vivo, por 72 horas después de la infección, las vacunas indujeron IFN y la expresión de genes estimulada por IFN, con menor inducción de citocinas proinflamatorias. Para varios genes (TLR3, IFNc, OASL, Mx1, NOS2A e IL-1ß), los efectos sobre la expresión de genes fueron aditivos para la infección por HVT, SB1 y HVT/SB1. Se Observaron patrones de inducción similares en embriones de pollo y pollos de engorde comerciales vacunados in ovo con HVT/SB-1 o con la combinación bivalente recombinante basada en HVT (HVT-LT/SB-1). Además, la vacunación in ovo con HVT/SB-1 o HVT-LT/SB-1 parecen acelerar la maduración inmune, con patrones de expresión que sugieren una migración acelerada de células T y células asesinas naturales en el bazo. Finalmente, la vacuna HVT/SB-1 dio como resultado una inducción coordinada de IL-12p40 y una regulación a la baja de supresores de las señales de citocinas 1 y 3, indicativas de los patrones clásicos de macrófagos 1 y células cooperadoras tipo 1.