Haji Akbar

Marek’s disease virus (MDV) is an oncogenic herpesvirus that establishes latency in CD4⁺ T cells, from which it reactivates to initiate productive replication and dissemination. To define host mechanisms governing the transition from latency to early and late lytic replication, we developed recombinant MDV expressing early RLORF4mRFP and late UL47eGFP, which was used to isolate latent (Lo; mRFP low), early lytic (Hi; mRFP high), and late lytic (DP; mRFP⁺eGFP⁺) populations from MDV-induced lymphoblastoid cell lines (LCLs). Two independent LCLs (Lines 62 and 82) were subjected to Illumina RNA sequencing after sorting-purified populations. Viral transcription increased progressively from Lo to Hi to DP populations; however, initiation of viral gene expression differed markedly between cell lines. During the early transition (Lo to Hi), Line 82 exhibited robust induction of 17 viral genes, whereas Line 62 showed only two differentially expressed viral genes. However, both lines converged on a highly conserved transcriptional program during the transition from latency (Lo) to late-lytic replication (DP), sharing 118 viral transcripts, most of which were structural and assembly-associated genes. Among the host transcriptional responses, Line 82 exhibited activation of TP53-associated stress signaling, chromatin remodeling factors, and RNA biogenesis pathways, consistent with a permissive cellular state. In contrast, Line 62 displayed enhanced inflammatory, metabolic, and proteostasis-associated signatures, suggesting a restrictive environment that limits early viral induction. Collectively, these findings demonstrate that MDV reactivation is a host-gated process in which the cellular state governs the initiation of the lytic switch, whereas downstream replication proceeds through a conserved viral program. IMPORTANCE: This study provides critical insights into the host-virus dynamics governing herpesvirus reactivation from latency. By developing a dual-reporter system using recombinant Marek’s disease virus (MDV) in MDV-transformed lymphoblastoid cell lines (LCLs), an oncogenic herpesvirus model, reactivating cells were sorted for latent, early-lytic, and late-lytic populations from two independent LCLs. The initial transition from latency to early-lytic replication was highly variable and strongly influenced by cellular state: one line showed rapid viral gene induction linked to stress, chromatin remodeling, and permissive conditions, while the other exhibited a more restrictive, inflammatory/metabolic response. However, progression to late-lytic replication converged on a highly conserved viral transcriptional program dominated by structural genes. These findings establish that host cellular context gates the lytic switch in herpesvirus reactivation, offering a powerful framework for understanding latency control across oncogenic herpesviruses and potential therapeutic targets to prevent reactivation and tumor formation.

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.

The conserved herpesvirus protein kinase (CHPK) is encoded by all members of the Orthoherpesviridae and contributes to replication in cell culture but is not strictly required. Marek’s disease virus (MDV) CHPK is dispensable for replication in cultured cells yet essential for horizontal transmission in chickens. To elucidate its role during natural infection, we performed RNA sequencing (RNA-seq) and mass spectrometry (MS)-based phosphoproteomics on spleen and feather follicle epithelial skin cells from chickens infected with wild-type or CHPK-null MDV. RNA-seq detected only a limited number of viral transcripts in the spleen—including latency-associated transcripts (LATs) and the major oncogene Meq—with minimal differences between wild-type and CHPK-null infections. In feather follicle epithelial skin cells, the full repertoire of viral genes was expressed, but only seven genes showed differential expression between wild-type and CHPK-null viruses. In striking contrast, MS-based phosphoproteomics identified many differentially phosphorylated proteins, including 21 viral proteins. These findings indicate that CHPK’s critical functions in skin replication and subsequent horizontal transmission are primarily mediated through post-translational modifications (PTMs) rather than transcriptional regulation. Among the CHPK-targeted viral proteins were three MDV-unique proteins, eight conserved within the Alphaherpesvirinae , and ten conserved across the Orthoherpesviridae . In silico analysis revealed that many differentially phosphorylated serine and threonine residues lie near or within predicted nuclear localization signals (NLS) and nuclear export signals (NES). Functional validation confirmed that several of these motifs actively control nucleocytoplasmic shuttling of the respective viral proteins. Collectively, these data suggest that MDV CHPK orchestrates the subcellular localization of multiple viral proteins in epithelial skin cells via phosphorylation, thereby enabling efficient replication and horizontal transmission in the natural host. AUTHOR SUMMARY: Understanding the mechanisms by which herpesviruses replicate and spread within their natural hosts and identifying the viral genes essential for these processes are fundamental to developing effective antiviral strategies. Marek’s disease virus (MDV), a highly contagious alphaherpesvirus, remains a major economic threat to the global poultry industry while serving as a powerful natural animal model for studying herpesvirus pathogenesis and transmission in vivo . Using an established in vivo enrichment method for infected cells, we conducted a comprehensive analysis of viral gene expression, protein abundance, and post-translational modifications (PTMs) during natural infection. Remarkably, RNA sequencing revealed virtually no differences in viral transcription between wild-type and CHPK-null viruses in either spleen or feather follicle epithelial skin cells. In contrast, phosphoproteomics showed that CHPK extensively regulates the phosphorylation of multiple viral proteins specifically in skin epithelial cells. In silico and functional analyses further indicate that these CHPK-mediated phosphorylations occur near or within nuclear localization (NLS) and nuclear export (NES) signals, directly controlling the nucleocytoplasmic shuttling of key viral proteins. This work suggests CHPK as a master regulator of viral protein subcellular localization during replication in the natural host and highlights CHPK orthologs as promising broad-spectrum therapeutic targets against herpesviruses.

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.

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.