Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to a lack of natural virus-host model systems. Marek’s disease is a devastating herpesviral disease of chickens caused by Marek’s disease virus (MDV) and an excellent natural model to study skin-tropic herpesviruses and transmission. Like varicella zoster virus that causes chicken pox in humans, the only site where infectious cell-free MD virions are efficiently produced is in epithelial skin cells, a requirement for host-to-host transmission. Here, we enriched for heavily infected feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined short- and long-read RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via IsoSeq transcripts, short-read intron-spanning sequencing reads, and a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5’ ends of two core herpesviral genes, pUL47 and ICP4, along with strong evidence of independent transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems. In the natural host, the transcriptome and proteome of many herpesviruses are poorly defined. Here, we evaluated the viral transcriptome and proteome in feather follicle epithelial skin cells of chickens infected with Marek’s disease virus (MDV), an important poultry pathogen as well as an excellent model for skin-tropic human alphaherpesvirus replication in skin cells. Using fluorescently tagged virus, we significantly enriched the number of infected cells sampled from live chickens, greatly enhancing the detection of viral transcripts and proteins within a host background. We carefully defined the in vivo transcriptome using both short-read RNA-Seq and long-read full-length transcript sequencing, and deep MS/MS-based proteomics from the enriched virus-infected feather follicle samples confirmed the translation of most transcripts and identified novel expressed peptides supportive of an increasingly complex translational and regulatory viral landscape. The demonstrated deep peptide sequencing capability can serve as a template for future in vivo work in herpesviral proteomics.

Conserved Herpesviridae protein kinases (CHPK) are conserved among all members of the Herpesviridae. Herpesviruses lacking CHPK propagate in cell culture at varying degrees, depending on the virus and cell culture system. CHPK is dispensable for Marek’s disease herpesvirus (MDV) replication in cell culture and experimental infection in chickens; however, CHPK—particularly its kinase activity—is essential for horizontal transmission in chickens, also known as natural infection. To address the importance of CHPK during natural infection in chickens, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics of samples collected from live chickens. Comparing modification of viral proteins in feather follicle epithelial (FFE) cells infected with wildtype or a CHPK-null virus, we identified the US10 protein (pUS10) as a potential target for CHPK in vivo. When expression of pUS10 was evaluated in cell culture and in FFE skin cells during in vivo infection, pUS10 was severely reduced or abrogated in cells infected with CHPK mutant or CHPK-null viruses, respectively, indicating a potential role for pUS10 in transmission. To test this hypothesis, US10 was deleted from the MDV genome, and the reconstituted virus was tested for replication, horizontal transmission, and disease induction. Our results showed that removal of US10 had no effect on the ability of MDV to transmit in experimentally infected chickens, but disease induction in naturally infected chickens was significantly reduced. These results show CHPK is necessary for pUS10 expression both in cell culture and in the host, and pUS10 is important for disease induction during natural infection.

Conserved herpesvirus protein kinases (CHPK) are conserved among the Orthoherpesviridae. Marek's disease virus (MDV) CHPK is dispensable for replication in chicken splenocytes but essential for replication in epithelial skin cells, where the virus is shed. We hypothesized that 1) MDV alters cellular gene expression in both tissues, 2) the responses differ between the spleen and skin, and 3) CHPK regulates these responses. To test these hypotheses, we collected spleen and epithelial skin tissues from chickens infected with wild-type MDV (vCHPKwt) or a CHPK-null MDV (vΔCHPK). RNA sequencing revealed significant differences in cellular gene expression between mock- and vCHPKwt-infected tissues. Pronounced differences were noted in the spleen, while the skin exhibited minimal changes. Functional enrichment analysis revealed that vCHPKwt infection significantly impacted the immune response, membrane transport, cell cycle regulation, and metabolic processes in both tissues. Comparisons between vΔCHPK and vCHPKwt in the spleen showed alterations in cellular interactions and immune system pathways. Global and phosphoproteomic profiling using mass spectrometry in the skin uncovered significant post-translational modifications of immune and energy regulators. Our findings demonstrate that CHPK plays a vital role in regulating cellular processes in the spleen at the transcriptional level while having a lesser impact on gene transcription in skin cells. Instead, CHPK influences cellular pathways post-transcriptionally, likely through protein phosphorylation. This study provides important insights into the mechanistic significance of CHPK during herpes viral replication in the natural host and during horizontal transmission.

Heat stress is a major challenge to livestock production; affecting physiological homeostasis, metabolic function, and productivity. Although goats possess adaptive mechanisms to cope with elevated temperatures, prolonged heat exposure can compromise their health and performance. Antioxidant supplementation has been proposed as a nutritional strategy to mitigate the negative effects of heat stress; however, information on the efficacy of plant-derived antioxidants such as Artemisia absinthium remains limited. Therefore, this study evaluated the effects of ascorbic acid (ASC; vitamin C), Artemisia absinthium (ART; wormwood), and their combination on thermoregulatory, hematobiochemical, and gene expression responses in heat-stressed Spanish goats. Thirty-two adult female goats (4–5 years old; 48.8 ± 8.1 kg) were stratified by body weight and randomly assigned to four treatments (n = 8 per treatment): control (CON; no supplement), ASC (2 g/goat/day), ART (2 g/goat/day), and ASC+ART (1 g each/goat/day), and supplemented for 42 days under summer grazing conditions. Ambient temperature and relative humidity were recorded hourly to calculate the temperature–humidity index (THI). Rectal temperature (RT), respiratory rate (RR), and coat surface temperature (CST) were measured weekly. Blood samples were analyzed for packed cell volume, albumin, total protein, blood urea nitrogen, cholesterol, triglycerides, creatinine, thyroxine, and glucose. Peripheral blood mononuclear cells were isolated for gene expression analysis of HSP70, CAT, and GPX1 by quantitative PCR. Heat production was measured in a subset of goats using respiratory calorimetry. Physiological and blood data were analyzed using the MIXED procedure of SAS, whereas gene expression data were analyzed using one-way ANOVA. Mean THI, ambient temperature, and relative humidity during the study were 78.0 ± 0.44, 28.7 ± 0.69 °C, and 62.9 ± 1.08%, respectively, confirming sustained heat stress conditions. Rectal temperature was significantly higher in the ASC+ART group than in the other treatments (p < 0.05). Respiratory rate also differed among treatments (p < 0.01), with higher values in the ART and ASC+ART groups. The coefficient of heat tolerance was significantly affected by treatment (P = 0.033), and the ASC+ART group had the lowest value. Heat production per unit metabolic body weight was significantly lower in the ART group than in the other groups (p < 0.05). Triglyceride concentrations were significantly lower, whereas glucose concentration was significantly higher, in the ART group (p < 0.05). Expression of GPX1, CAT and HSP70 was significantly lower in supplemented groups than in the control group, indicating reduced oxidative stress. In conclusion, individual supplementation with Artemisia absinthium improved metabolic efficiency and favorably altered selected biochemical and molecular responses under heat stress, whereas ascorbic acid had limited effects. The combined treatment did not improve thermoregulation and appeared less favorable than Artemisia absinthium alone.

Herpesvirus infections, including herpes simplex virus (HSV), Epstein–Barr virus (EBV), and cytomegalovirus (CMV), present significant challenges in diagnosis, treatment, and transmission control. Despite advances in medical technology, managing these infections remains complex due to the viruses’ ability to establish latency and their widespread prevalence. Artificial Intelligence (AI) has emerged as a transformative tool in biomedical science, enhancing our ability to understand, predict, and manage infectious diseases. In veterinary virology, AI applications offer considerable potential for improving diagnostics, forecasting outbreaks, and implementing targeted control strategies. This review explores the growing role of AI in advancing our understanding of herpesvirus infection, particularly those caused by MDV, through improved detection, transmission modeling, treatment strategies, and predictive tools. Employing AI technologies such as machine learning (ML), deep learning (DL), and natural language processing (NLP), researchers have made significant progress in addressing diagnostic limitations, modeling transmission dynamics, and identifying potential therapeutics. Furthermore, AI holds the potential to revolutionize personalized medicine, predictive analytics, and vaccine development for herpesvirus-related diseases. The review concludes by discussing ethical considerations, implementation challenges, and future research directions necessary to fully integrate AI into clinical and veterinary practice.

Herpesvirus infections, including herpes simplex virus (HSV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV), present significant challenges in diagnosis, treatment, and transmission control. Despite advances in medical technology, managing these infections remains complex due to the viruses' ability to establish latency and their widespread prevalence. Artificial Intelligence (AI) has emerged as a transformative tool in biomedical science, enhancing our ability to understand, predict, and manage infectious diseases. In veterinary virology, AI applications offer considerable potential for improving diagnostics, forecasting outbreaks, and implementing targeted control strategies. This review explores the growing role of AI in advancing our understanding of herpesvirus infection, particularly those caused by MDV, through improved detection, transmission modeling, treatment strategies, and predictive tools. Employing AI technologies such as machine learning (ML), deep learning (DL), and natural language processing (NLP), researchers have made significant progress in addressing diagnostic limitations, modeling transmission dynamics, and identifying potential therapeutics. Furthermore, AI holds the potential to revolutionize personalized medicine, predictive analytics, and vaccine development for herpesvirus-related diseases. The review concludes by discussing ethical considerations, implementation challenges, and future research directions necessary to fully integrate AI into clinical and veterinary practice.