ICP4 Gene Research Articles

Co-Regulation Mechanism of Host p53 and Fos in Transcriptional Activation of ILTV Immediate-Early Gene ICP4.

Infectious laryngotracheitis virus (ILTV) exhibits a cascade expression pattern of encoded genes, and ICP4 is the only immediate-early gene of ILTV, which plays a crucial role in initiating the subsequent viral genes. Therefore, studying the transcriptional regulation mechanism of ICP4 holds promise for effectively blocking ILTV infection and spread. Host transcriptional factors p53 and Fos are proven to regulate a variety of viral infections, and our previous studies have demonstrated their synergistic effects in regulating ILTV infection. In this study, we constructed eukaryotic expression vectors for p53 and Fos as well as their specific siRNAs and transfected them into a chicken hepatoma cell line. The results showed that knocking down p53 or Fos significantly inhibited ICP4 transcription, while overexpressing p53 or Fos had an opposite effect. A further CoIP and ChIP-qPCR assay suggested p53 and Fos physically interacted with each other, and jointly bound to the upstream transcriptional regulatory region of ICP4. To elucidate the specific mechanisms of p53 and Fos in regulating ICP4 transcription, we designed p53 and Fos protein mutants by mutating their DNA binding domains, which significantly reduced their binding ability to DNA without affecting their interaction. The results showed that Fos directly bound to the promoter region of ICP4 as a binding target of p53, and the p53-Fos protein complex acted as a transcriptional co-regulator of ICP4. Studying the transcriptional process and regulatory pattern of ICP4 is of great significance for understanding the molecular mechanism of ILTV infection, and thus for finding effective methods to control and prevent it.

Suppression of HSV-1 infection and viral reactivation by CRISPR-Cas9 gene editing in 2D and 3D culture models

Although our understanding of HSV-1 biology has been considerably enhanced, developing therapeutic strategies to eliminate HSV-1 in latently infected individuals remains a public health concern. Current antiviral drugs used for the treatment of HSV-1 complications are not specific and do not address latent infection. We recently developed a CRISPR/Cas9-based gene editing platform to specifically target the HSV-1 genome. In this study, we further used 2D Vero cell culture and 3D hiPSC-derived cerebral organoid (CO) models to assess the effectiveness of our editing constructs targeting viral ICP0 or ICP27 gene. We found that targeting the ICP0 or ICP27 gene with AAV2-CRISPR/Cas9 vectors in Vero cells drastically suppressed HSV-1 replication. In addition, we productively infected COs with HSV-1, characterized the viral replication kinetics, and established a viral latency model. Finally, we discovered that ICP0- or ICP27-targeting AAV2-CRISPR/Cas9 vector significantly reduced viral rebound in the COs that were latently infected with HSV-1. In summary, our results suggest that CRISPR/Cas9 gene editing of HSV-1 is an efficient therapeutic approach to eliminate the latent viral reservoir and treat HSV-1 associated complications.

Molecular characterization of Marek's Disease virus reveals reticuloendotheliosis virus-long terminal repeat integration in the genome of the field isolates in Egypt

The highly contagious, immunosuppressive, and cancer-causing Marek's disease virus (MDV) infects chickens. The financial costs of Marek's disease (MD) are significant for the chicken industry. In this study, a total of 180 samples from chicken farms suspected to be MDV-infected were collected. The chickens were sampled during the period between the months of October 2016 and February 2018 at Dakahlia and Damietta Governorates, Egypt. A total of 36 pooled samples were created. The prepared samples were inoculated into embryonated chicken eggs (ECEs). Indirect fluorescent antibody technique (IFAT) and ICP4 gene-based polymerase chain reaction (PCR) were used for MDV identification. For the genetic characterization of the identified virus, The ICP4 gene sequence was identified and compared with the sequences available from various regions of the world. Furthermore, the genomes of all detected MDVs were screened for the long terminal repeat (LTR) region of reticuloendotheliosis (REV) in their genomes. The results showed that 31 out of 36 pooled samples (86.1%) inoculated into ECEs displayed the characteristic pock lesions. By using IFAT and PCR to identify MDV in ECEs, positive results were found in 27 samples (75%). The Egyptian virus is thought to be genetically closely related to MDVs circulating in Ethiopia, China, and India. REV-LTR was amplified from 6 out of 27 field isolates genomes (22.2 %) while MDV vaccine strains were free from REV-LTR insertion. The integrated REV-LTRs depicted a close genetic relationship with those integrated in fowl poxvirus (FWPV) circulating in Egypt as well as those integrated in FWPVs and MDVs from China, USA, South Africa, and Australia. To the best of our knowledge, this investigation represents the first identification and characterization of REV-LTR insertions in Egyptian MDV field isolates. Given the findings above, additional research in the future seems crucial to determine how the REV-LTR insertions affect MDV pathogenesis, virulence, and insufficient vaccination protection.

A Revision of Herpes Simplex Virus Type 1 Transcription: First, Repress; Then, Express.

The herpes virus genome bears more than 80 strong transcriptional promoters. Upon entry into the host cell nucleus, these genes are transcribed in an orderly manner, producing five immediate-early (IE) gene products, including ICP0, ICP4, and ICP22, while non-IE genes are mostly silent. The IE gene products are necessary for the transcription of temporal classes following sequentially as early, leaky late, and true late. A recent analysis using precision nuclear run-on followed by deep sequencing (PRO-seq) has revealed an important step preceding all HSV-1 transcription. Specifically, the immediate-early proteins ICP4 and ICP0 enter the cell with the incoming genome to help preclude the nascent antisense, intergenic, and sense transcription of all viral genes. VP16, which is also delivered into the nucleus upon entry, almost immediately reverses this repression on IE genes. The resulting de novo expression of ICP4 and ICP22 further repress antisense, intergenic, and early and late viral gene transcription through different mechanisms before the sequential de-repression of these gene classes later in infection. This early repression, termed transient immediate-early protein-mediated repression (TIEMR), precludes unproductive, antisense, intergenic, and late gene transcription early in infection to ensure the efficient and orderly progression of the viral cascade.

Characterization of infectious laryngotracheitis virus isolated from commercial layer chickens in Bangladesh during the year 2021-2022.

Infectious laryngotracheitis virus (ILTV) is responsible for causing infectious laryngotracheitis (ILT), which is a rapidly spreading and extremely transmissible disease in chickens. The current research aims to isolate and characterize ILTV from layer chickens in Bangladesh. A total of 345 samples (trachea, larynx, and lungs) were collected from ILT-suspected dead and sick layer chickens of 32 ILT-suspected farms in three different outbreak districts (Gazipur, Tangail, and Mymensingh) of Bangladesh during the outbreak year 2021-2022. Rapid detection kits examined the samples for avian influenza virus (AIV) and Newcastle disease virus (NDV). ILTV-specific primers were used to screen 72 NDV- and AIV-negative samples by polymerase chain reaction (PCR). Using chorioallantoic membrane (CAM), the study isolated the ILT virus from 9 to 10-day-old seronegative embryonated chicken eggs (ECEs) using selected PCR-positive samples. The virus was confirmed using nucleotide sequencing, agar gel immunodiffusion test (AGIDT), viral neutralization test (VNT), and pathogenicity evaluations using mortality index for chicken embryos (MICEs) and intra-tracheal pathogenicity index (ITPI). The results indicated that among the PCR-positive 10 samples, only two (Alim_ILT_1001 and Alim_ILT_1,000) were found positive using ECEs. There were two field isolates of ILTVs, as shown by the amplicon size of the ICP4 gene-based PCR. A phylogenetic study of the ICP4 gene revealed that the recent isolates have a close similarity with the ILTV isolates of Turkey, Bangladesh, and Australia. AGIDT revealed strong precipitation lines due to ILTV-specific antibodies reacting with field viruses, while VNT neutralized both isolates with conventional ILTV antibodies. The pathogenicity testing indicated that Alim_ILT_1001 had MICE and ITPI values of 0.77 and 0.63, whereas Alim_ILT_1,000 had 0.71 and 0.57. Both the ILTV isolates have similarities with the isolates of Turkey, Bangladesh, and Australia, and they are highly virulent for chickens.

ICP4-Associated Activation of Rap1b Facilitates Herpes Simplex Virus Type I (HSV-1) Infection in Human Corneal Epithelial Cells.

The strong contribution of RAS-related protein 1b (Rap1b) to cytoskeleton remodeling determines intracellular and extracellular physiological activities, including the successful infection of viruses in permissive cells, but its role in the HSV-1 life cycle is still unclear. Here, we demonstrated that the HSV-1 immediate early (IE) gene ICP4 inhibits protein kinase A (PKA) phosphorylation to induce Rap1b-activation-mediated viral infection. Rap1b activation and membrane enrichment begin at the early stage of HSV-1 infection and remain active during the proliferation period of the virus. Treating the cells with Rap1b small interfering RNA (siRNA) showed a dose-dependent decrease in viral infection levels, but no dose-dependent increase was observed after Rap1b overexpression. Further investigation indicated that the suppression of Rap1b activation derives from phosphorylated PKA and Rap1b mutants with partial or complete prenylation instead of phosphorylation, which promoted viral infection in a dose-dependent manner. Furthermore, the PKA agonist Forskolin disturbed Rap1b activation in a dose-dependent manner, accompanied by a decreasing trend in viral infection. Moreover, the HSV-1 IE gene ICP4 induced PKA dephosphorylation, leading to continuous Rap1b activation, followed by cytoskeleton rearrangement induced by cell division control protein 42 (CDC42) and Ras-related C3 botulinum toxin substrate 1 (RAC1). These further stimulated membrane-triggered physiological processes favoring virus infection. Altogether, we show the significance of Rap1b during HSV-1 infection and uncover the viral infection mechanism determined by the posttranslational regulation of the viral ICP4 gene and Rap1b host protein.

Herpes Simplex Virus-1 ICP27 Nuclear Export Signal Mutants Exhibit Cell Type-Dependent Deficits in Replication and ICP4 Expression.

Herpes simplex virus type-1 (HSV-1) protein ICP27 is an essential immediate early (IE) protein that promotes the expression of viral early (E) and late (L) genes via multiple mechanisms. Our understanding of this complex regulatory protein has been greatly enhanced by the characterization of HSV-1 mutants bearing engineered alterations in the ICP27 gene. However, much of this analysis has been performed in interferon-deficient Vero monkey cells. Here, we assessed the replication of a panel of ICP27 mutants in several other cell types. Our analysis shows that mutants lacking ICP27's amino (N)-terminal nuclear export signal (NES) display a striking cell type-dependent growth phenotype, i.e., they grow semi-permissively in Vero and some other cells but are tightly blocked for replication in primary human fibroblasts and multiple human cell lines. This tight growth defect correlates with a failure of these mutants to replicate viral DNA. We also report that HSV-1 NES mutants are deficient in expressing the IE protein ICP4 at early times postinfection. Analysis of viral RNA levels suggests that this phenotype is due, at least in part, to a defect in the export of ICP4 mRNA to the cytoplasm. In combination, our results (i) show that ICP27's NES is critically important for HSV-1 replication in many human cells, and (ii) suggest that ICP27 plays a heretofore unappreciated role in the expression of ICP4. IMPORTANCE HSV-1 IE proteins drive productive HSV-1 replication. The major paradigm of IE gene induction, developed over many years, involves the parallel activation of the five IE genes by the viral tegument protein VP16, which recruits the host RNA polymerase II (RNAP II) to the IE gene promoters. Here, we provide evidence that ICP27 can enhance ICP4 expression early in infection. Because ICP4 is required for transcription of viral E and L genes, this finding may be relevant to understanding how HSV-1 enters and exits the latent state in neurons.

CLEAR Strategy Inhibited HSV Proliferation Using Viral Vectors Delivered CRISPR-Cas9.

Herpes simplex virus type 1 (HSV-1) is a leading cause of encephalitis and infectious blindness. The commonly used clinical therapeutic drugs are nucleoside analogues such as acyclovir. However, current drugs for HSV cannot eliminate the latent virus or viral reactivation. Therefore, the development of new treatment strategies against latent HSV has become an urgent need. To comprehensively suppress the proliferation of HSV, we designed the CLEAR strategy (coordinated lifecycle elimination against viral replication). VP16, ICP27, ICP4, and gD-which are crucial genes that perform significant functions in different stages of the HSV infection lifecycle-were selected as targeting sites based on CRISPR-Cas9 editing system. In vitro and in vivo investigations revealed that genome editing by VP16, ICP27, ICP4 or gD single gene targeting could effectively inhibit HSV replication. Moreover, the combined administration method (termed "Cocktail") showed superior effects compared to single gene editing, which resulted in the greatest decrease in viral proliferation. Lentivirus-delivered CRISPR-Cas9/gRNA editing could effectively block HSV replication. The CLEAR strategy may provide new insights into the potential treatment of refractory HSV-1-associated diseases, particularly when conventional approaches have encountered resistance.

Marek’s disease in chicken farms from Northwest Ethiopia: gross pathology, virus isolation, and molecular characterization

Marek’s disease virus (MDV) is a highly contagious, immunosuppressive, and oncogenic chicken pathogen causing marek’s disease (MD). In this outbreak-based study, 70 dual-purpose chickens that originated from poultry farms in Northwest Ethiopia and suspected of MD were sampled for pathological and virological study from January 2020 to June 2020. Clinically, affected chickens showed inappetence, dyspnea, depression, shrunken combs, and paralysis of legs, wings, and neck, and death. Pathologically, single or multiple greyish white to yellow tumor-like nodular lesions of various size were appreciated in visceral organs. In addition, splenomegaly, hepatomegaly, renomegaly, and sciatic nerve enlargement were observed. Twenty-seven (27) pooled clinical samples i.e. 7 pooled spleen samples and 20 pooled feathers samples were aseptically collected. Confluent monolayer of Chicken Embryo Fibroblast cells was inoculated with a suspension of pathological samples. Of this, MDV-suggestive cytopathic effects were recorded in 5 (71.42%) and 17 (85%) pooled spleen and feather samples respectively. Molecular confirmation of pathogenic MDV was conducted using conventional PCR amplifying 318 bp of ICP4 gene of MDV-1, of which, 40.9% (9/22) tested positive. In addition, 5 PCR-positive samples from various farms were sequenced further confirming the identity of MDV. The ICP4 partial gene sequences were submitted to GenBank with the following accession numbers: OP485106, OP485107, OP485108, OP485109, and OP485110. Comparative phylogenetics showed, two of the isolates from the same site, Metema, seem to be clonal complexes forming distinct cluster. The other three isolates, two from Merawi and one from Debretabor, appear to represent distinct genotypes although the isolate from Debretabor is closer to the Metema clonal complex. On the other hand, the isolates from Merawi appeared genetically far related to the rest of the 3 isolates and clustered with Indian MDV strains included in the analysis. This study presented the first molecular evidence of MDV in chicken farms from Northwest Ethiopia. Biosecurity measures should strictly be implemented to hinder the spread of the virus. Nationwide studies on molecular characteristics of MDV isolates, their pathotypes, and estimation of the economic impact associated with the disease may help justify production and use of MD vaccines within the country.

Inhibitors of One or More Cellular Aurora Kinases Impair the Replication of Herpes Simplex Virus 1 and Other DNA and RNA Viruses with Diverse Genomes and Life Cycles.

We utilized a high-throughput cell-based assay to screen several chemical libraries for inhibitors of herpes simplex virus 1 (HSV-1) gene expression. From this screen, four aurora kinase inhibitors were identified that potently reduced gene expression during HSV-1 lytic infection. HSV-1 is known to interact with cellular kinases to regulate gene expression by modulating the phosphorylation and/or activities of viral and cellular proteins. To date, the role of aurora kinases in HSV-1 lytic infection has not been reported. We demonstrated that three aurora kinase inhibitors strongly reduced the transcript levels of immediate-early (IE) genes ICP0, ICP4, and ICP27 and impaired HSV-1 protein expression from all classes of HSV-1, including ICP0, ICP4, ICP8, and gC. These restrictions caused by the aurora kinase inhibitors led to potent reductions in HSV-1 viral replication. The compounds TAK 901, JNJ 7706621, and PF 03814735 decreased HSV-1 titers by 4,500-, 13,200-, and 8,400-fold, respectively, when present in a low micromolar range. The antiviral activity of these compounds correlated with an apparent decrease in histone H3 phosphorylation at serine 10 (H3S10ph) during viral infection, suggesting that the phosphorylation status of H3 influences HSV-1 gene expression. Furthermore, we demonstrated that the aurora kinase inhibitors also impaired the replication of other RNA and DNA viruses. These inhibitors significantly reduced yields of vaccinia virus (a poxvirus, double-stranded DNA, cytoplasmic replication) and mouse hepatitis virus (a coronavirus, positive-sense single-strand RNA [ssRNA]), whereas vesicular stomatitis virus (rhabdovirus, negative-sense ssRNA) yields were unaffected. These results indicated that the activities of aurora kinases play pivotal roles in the life cycles of diverse viruses. IMPORTANCE We have demonstrated that aurora kinases play a role during HSV-1 lytic infection. Three aurora kinase inhibitors significantly impaired HSV-1 immediate-early gene expression. This led to a potent reduction in HSV-1 protein expression and viral replication. Together, our results illustrate a novel role for aurora kinases in the HSV-1 lytic cycle and demonstrate that aurora kinase inhibitors can restrict HSV-1 replication. Furthermore, these aurora kinase inhibitors also reduced the replication of murine coronavirus and vaccinia virus, suggesting that multiple viral families utilize the aurora kinases for their own replication.

Immediate Early Proteins of Herpes Simplex Virus Transiently Repress Viral Transcription before Subsequent Activation.

Herpes simplex virus 1 (HSV-1) utilizes cellular RNA polymerase II (Pol) to transcribe its genes in one of two phases. In the latent phase, viral transcription is highly restricted, but during the productive lytic phase, more than 80 genes are expressed in a temporally coordinated cascade. In this study, we used Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to characterize early viral transcriptional events using HSV-1 immediate early (IE) gene mutants, corresponding genetically repaired viruses, and wild-type virus. Unexpectedly, in the absence of the IE genes ICP4, ICP22, and ICP0 at 1.5 hours postinfection (hpi), we observed high levels of aberrant transcriptional activity across the mutant viral genomes but substantially less on either wild-type or the congenic repaired virus genomes. This feature was particularly prominent in the absence of ICP4 expression. Cycloheximide treatment during infection with both the ICP4 and ICP22 mutants and their respective genetic repairs did not alter the relative distribution of Pol activity, but it increased overall activity across both viral genomes, indicating that both virion components and at least some de novo protein synthesis were required for full repression. Overall, these data reveal that prior to their role in transcriptional activation, IE gene products and virion components first repress transcription and that the HSV-1 lytic transcriptional cascade is mediated through subsequent derepression steps. IMPORTANCE HSV-1 transcription during productive replication is believed to comprise a series of activation steps leading to a specific sequence of gene expression. Here, we show that virion components and IE gene products ICP0, ICP4, and ICP22 first repress viral gene transcription to various degrees before subsequently activating specific gene subsets. It follows that the entire HSV transcriptional program involves a series of steps to sequentially reverse this repression. This previously uncharacterized repressive activity of IE genes very early in infection may represent an important checkpoint allowing HSV-1 to orchestrate either the robust lytic transcriptional cascade or the more restricted transcriptional program during latency.

Detection and molecular characterization of infectious laryngotracheitis virus (ILTV) in chicken with respiratory signs in Brazil during 2015 and 2016.

Avian infectious laryngotracheitis (ILT) is a respiratory disease that causes severe economic losses in the poultry industry, mainly due to high morbidity and mortality and reduced egg production. Molecular characterization was performed on samples collected from flocks in the Brazilian States of São Paulo, Pernambuco, and Minas Gerais during 2015 and 2016 that presented clinical signs of respiratory disease. End-point PCR was used for viral detection, and DNA sequencing was used for differentiation of vaccine and field strains. Molecular analysis based on the infected cell protein (ICP4) gene separated four of the nine samples together with previous Brazilian isolates (São Paulo and Minas Gerais), one sample was grouped on the same branch as Minas Gerais strains (along with another related sample), one sample was separately branched but still related to the tissue culture origin (TCO) vaccine strain, and two samples were grouped on the same branch as the TCO vaccine strain. Molecular analysis of the thymidine kinase (TK) gene showed the existence of strains of both high and low virulence. The characterization of two fragments of the ICP4 gene and a fragment of the TK gene in this study suggested that the virus circulating in Guatapará, as well as those in Barretos and Itanhandu, that is causing respiratory problems in birds is a highly virulent field strain. The clinical signs point to a TCO vaccine strain that most likely underwent some reversal event and is a latent reactivated infection.

Molecular and genetic detection of infectious laryngeotrachitis disease virus in broiler farms after a disease outbreak in Egypt.

Infectious laryngotracheitis (ILT) is a viral respiratory illness in poultry that causes massive financial losses. This research aimed to isolate and identify the ILT virus in suspected outbreaks of broiler flocks in Egypt during 2020-2021, besides investigating its genetic link with other circulating strains. Real-time-PCR was used to test 57 samples taken from unvaccinated broiler farms. Ten samples are positive for ILTV, and the virus is being isolated in SPF chicken embryos. The Sanger sequencing was used to conduct (partial) sequencing of the infected cell protein4 gene (ICP4) for eight isolates. Phylogenetic analysis conducted Maximum Likelihood, comparative sequencing analysis of ICP4 of strains under study with vaccination ILT reference strains reveled that all isolates were clustered into two major groups. The (OM291843and OM291846) clustered together with the chicken embryo origin vaccine strains (IV and V group). The remaining six strains belong to the TCO vaccine(I, II and III group). The total sequence similarity between the strains under study and the various Egyptian strains varied from (97 to 100%) while the similarity with TCO or chicken embryo origin -vaccine strains ranged from (95to 100%). There were no deletions detected in the 272-283-bp region of the ICP4 gene. Detection of arginine to methionine substitutions at position 180 (R180M) and change of Serine to Asparagine at position 227 (S227N) in the (OM291843 and OM291846) which were previously described in chicken embryo origin -vaccine strains. This reveals that field strains may have evolved from vaccine strains, notably identification of non-synonymous substitutions which might be linked to the virulence strains' attenuation. Finally, independent of geographical distribution, both chicken embryo origin-vaccine-like and TCO-Vaccine-like virus strains were circulating in Egyptian non-vaccinated broiler flocks in 2020 and 2021. Despite their genetic differences, both viruses caused significant illnesses in the field.

Establishing a Herpesvirus Quiescent Infection in Differentiated Human Dorsal Root Ganglion Neuronal Cell Line Mediated by Micro-RNA Overexpression.

HSV-1 is a neurotropic pathogen associated with severe encephalitis, excruciating orofacial sensation, and other chronic neuropathic complications. After the acute infection, the virus may establish a lifelong latency in the neurons of trigeminal ganglia (TG) and other sensory and autonomic ganglia, including the dorsal root ganglia (DRG), etc. The reactivation occurred periodically by a variety of physical or emotional stressors. We have been developing a human DRG neuronal cell-culture model HD10.6, which mimics the mature neurons for latency and reactivation with robust neuronal physiology. We found that miR124 overexpression without acyclovir (ACV) could maintain the virus in a quiescent infection, with the accumulation of latency-associate transcript (LAT). The immediate-early (IE) gene ICP0, on the other hand, was very low and the latent viruses could be reactivated by trichostatin A (TSA) treatment. Together, these observations suggested a putative role of microRNA in promoting HSV-1 latency in human neurons.

Hypericin blocks the function of HSV-1 alkaline nuclease and suppresses viral replication

Ethnopharmacological relevanceHypericum perforatum L. has a long history in many countries of being used as a herbal medicine. It is also widely used in Chinese herbal medicine for the treatment of infections. Hypericin, a main component extracted from Hypericum perforatum L., has attracted the attention of many researchers for its remarkable antiviral, antitumor and antidepressant effects. Aim of the studyTo find plant molecules that inhibit the alkaline nuclease (AN) of herpes simplex virus type 1 (HSV-1) and suppress viral replication. Materials and methodsBioinformatics methods were used to determine which compounds from a variety of natural compounds in our laboratory interact with AN. By this means we predicted that hypericin may interact with AN and suppress HSV-1 replication. Experiments were then carried out to verify whether hypericin inhibits the bioactivity of AN. The Pichia pastoris expression system was used to obtain recombinant AN. The exonuclease and endonuclease activity of AN treated with hypericin were tested by electrophoresis. Immunohistochemical staining of the HSV-1 nucleocapsids was used to find out whether hypericin inhibits the intracellular function of AN. Real-time PCR and western blotting analysis were performed to test viral gene expression and viral protein synthesis. The extent of viral replication inhibited by hypericin was determined by a plaque assay and a time of addition assay. ResultsRecombinant AN was obtained by Pichia pastoris expression system. The exonuclease and endonuclease activity of recombinant AN were inhibited by hypericin in the electrophoresis assay. Hypericin showed no inhibitory effect on BeyoZonase™ Super Nuclease or DNase I. T5 Exonuclease activity was inhibited partially by10 μM hypericin, and was completely suppressed by 50 μM hypericin. Hind Ⅲ was inhibited by hypericin at concentrations greater than 100 μM, but EcoR I, BamH I, and Sal I were not inhibited by hypericin. HSV-1 nucleocapsids gathered in the nucleus when the viruses were treated with hypericin. Plaque formation was significantly reduced by hypericin (EC50 against HSV-1 F is 2.59 ± 0.08 μM and EC50 against HSV-1 SM44 is 2.94 ± 0.10 μM). UL12, ICP27, ICP8, gD, and UL53 gene expression (P < 0.01, 4.0 μM hypericin treated group vs control group) and ICP4 (P < 0.05, 6.0 μM hypericin treated group vs control group), ICP8 and gD (P < 0.05, 2.0 μM hypericin treated group vs control group) protein synthesis were inhibited by hypericin. In the time of addition assay, HSV-1 was suppressed by hypericin in the early stages of viral replication. Hypericin exhibits potent virucidal activity against HSV-1 and inhibits the adsorption and penetration of HSV-1. ConclusionHypericin inhibits the bioactivity of AN and suppresses HSV-1 replication. The data revealed a novel mechanism of the antiherpetic effect of hypericin.

Characterization of infectious laryngotracheitis virus isolates from laying hens during 2019-2020 outbreaks in Tamil Nadu, India.

Infectious laryngotracheitis (ILT) is an acute respiratory disease in chickens that is a serious threat to poultry-producing countries worldwide. In the present study, we isolated and characterized infectious laryngotracheitis (ILTV) virus isolates by sequencing and restriction fragment length polymorphism analysis of PCR-amplified products (PCR-RFLP). A total of 26 ILTV outbreaks were investigated that occurred between 2019 and 2020 in flocks that had not been vaccinated against ILTV. ILTV was isolated by cultivating tracheal samples in embryonated chicken eggs, which showed multiple opaque pock lesions and thickening of the chorioallantoic membrane after 120 hours of infection. The ILTV isolates were identified and characterized by PCR and sequencing a portion of the ICP4 and TK genes. Phylogenetic analysis based on the ICP4 region showed that the sequences clustered with chicken-embryo-origin vaccine-like strains. Sequence analysis of the ICP4 region differentiated chicken-embryo-origin (CEO), tissue-culture-origin (TCO), and field ILTV strains, with significant differences in nucleotide and amino acid sequences. Furthermore, PCR-RFLP analysis of the TK gene showed that the patterns were identical to those obtained with low-virulence and vaccine strains. In conclusion, sequencing of a portion of the ICP4 region of ILTV allowed differentiation of ILTV field, CEO, and TCO vaccine strains. In this study, CEO-vaccine-like strains were found to be the cause of ILTV outbreaks between 2019 and 2020 in Tamil Nadu in southern India.

Host MOV10 is induced to restrict herpes simplex virus 1 lytic infection by promoting type I interferon response.

Moloney leukemia virus 10 protein (MOV10) is an interferon (IFN)-inducible RNA helicase implicated in antiviral activity against RNA viruses, yet its role in herpesvirus infection has not been investigated. After corneal inoculation of mice with herpes simplex virus 1 (HSV-1), we observed strong upregulation of both MOV10 mRNA and protein in acutely infected mouse trigeminal ganglia. MOV10 suppressed HSV-1 replication in both neuronal and non-neuronal cells, and this suppression required the N-terminus, but not C-terminal helicase domain of MOV10. MOV10 repressed expression of the viral gene ICP0 in transfected cells, but suppressed HSV-1 replication independently of ICP0. MOV10 increased expression of type I IFN in HSV-1 infected cells with little effect on IFN downstream signaling. Treating the cells with IFN-α or an inhibitor of the IFN receptor eliminated MOV10 suppression of HSV-1 replication. MOV10 enhanced IFN production stimulated by cytoplasmic RNA rather than DNA. IKKε co-immunoprecipitated with MOV10 and was required for MOV10 restriction of HSV-1 replication. Mass spectrometry identified ICP27 as a viral protein interacting with MOV10. Co-immunoprecipitation results suggested that this interaction depended on the RGG box of ICP27 and both termini of MOV10. Overexpressed ICP27, but not its RGG-Box deletion mutant, rendered MOV10 unable to regulate HSV-1 replication and type I IFN production. In summary, MOV10 is induced to restrict HSV-1 lytic infection by promoting the type I IFN response through an IKKε-mediated RNA sensing pathway, and its activity is potentially antagonized by ICP27 in an RGG box dependent manner.

A surveillance for avian coronavirus infectious bronchitis virus, infectious laryngotracheitis virus, avian metapneumovirus, and avian reovirus in poultry flocks with respiratory signs in Türkiye

In this study, avian coronavirus infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (AMPV), and avian reovirus (ARV) were evaluated in broiler and layer flocks. For this purpose, tracheal swabs from 48 broiler and 45 layer flocks with respiratory signs were inoculated SPF embryonated chicken eggs for virus isolation. The viruses were identified by real-time PCR. Results showed that the most common virus in both broiler and layer farms was IBV with incidence rates of 58.33% and 46.67%, respectively. ILTV, AMPV, and ARV incidences in the samples were found to be 22.22%, 13.33%, and 4.44% in layer flocks while 2.08%, 8.33%, and 20.83% in broilers, respectively. The numbers of IBV+AMPV and IBV+ARV coinfections were 5 (11.11%) and 1 (2.22%) in layers, whereas, 1 (2.08%) and 5 (10.42%) broilers, respectively. In addition, 2 broiler flocks (4.17%) had triple infection with IBV, AMPV, and ARV. ILTV was detected always alone from the samples of layer and broiler flocks. Sequencing of S1 gene of selected IBV TR/L45 and TR/B42 isolates showed similarities with IS/1494/06 (HM131453) at the rates of 98.98% and 99.69%, respectively, while TR/L37, TR/L38, and TR/L39 isolates were identical to 4/91 (KF377577) vaccine strain at the rates of 99.01%, 99.01%, and 98.76%, respectively. Sequencing analysis of the ICP4 and TK genes of ILTV isolates revealed that they were all field strains with low virulence. The present data represent actual information on the genotypes of IBV and ILTV circulating in poultry flocks in Türkiye.

Triple‐mutated oncolytic herpes virus for treating both fast‐ and slow‐growing tumors

Oncolytic virus therapy has emerged as a promising treatment option against cancer. To date, oncolytic viruses have been developed for malignant tumors, but the need for this new therapeutic modality also exists for benign and slow‐growing tumors. G47∆ is an oncolytic herpes simplex virus type 1 (HSV‐1) with an enhanced replication capability highly selective to tumor cells due to genetically engineered, triple mutations in the γ34.5, ICP6 and α47 genes. To create a powerful, but safe oncolytic HSV‐1 that replicates efficiently in tumors regardless of growth speed, we used a bacterial artificial chromosome system that allows a desired promoter to regulate the expression of the ICP6 gene in the G47∆ backbone. Restoration of the ICP6 function in a tumor‐specific manner using the hTERT promoter led to a highly capable oncolytic HSV‐1. T‐hTERT was more efficacious in the slow‐growing OS‐RC‐2 and DU145 tumors than the control viruses, while retaining a high efficacy in the fast‐growing U87MG tumors. The safety features are also retained, as T‐hTERT proved safe when inoculated into the brain of HSV‐1 sensitive A/J mice. This new technology should facilitate the use of oncolytic HSV‐1 for all tumors irrespective of growth speed.

Fos Facilitates Gallid Alpha-Herpesvirus 1 Infection by Transcriptional Control of Host Metabolic Genes and Viral Immediate Early Gene.

Gallid alpha-herpesvirus 1, also known as avian infectious laryngotracheitis virus (ILTV), continues to cause huge economic losses to the poultry industry worldwide. Similar to that of other herpesvirus-encoded proteins, the expression of viral genes encoded by ILTV is regulated by a cascade, and the underlying regulatory mechanism remains largely unclear. The viral immediate-early (IE) gene ICP4 plays a prominent role in the initiation of the transcription of early and late genes during ILTV replication. In this study, we identified AP-1 as the key regulator of the transcription of ILTV genes by bioinformatics analysis of genome-wide transcriptome data. Subsequent functional studies of the key members of the AP-1 family revealed that Fos, but not Jun, regulates ILTV infection through AP-1 since knockdown of Fos, but not Jun, by gene silencing significantly reduced ICP4 transcription and subsequent viral genome replication and virion production. Using several approaches, we identified ICP4 as a bona fide target gene of Fos that regulated Fos and has Fos response elements within its promoter. Neither the physical binding of Jun to the promoter of ICP4 nor the transcriptional activity of Jun was observed. In addition, knockdown of Fos reduced the transcription of MDH1 and ATP5A1, genes encoding two host rate-limiting enzymes essential for the production of the TCA intermediates OAA and ATP. The biological significance of the transcriptional regulation of MDH1 and ATP5A1 by Fos in ILTV infection was supported by the fact that anaplerosis of OAA and ATP rescued both ICP4 transcription and virion production in infected cells under when Fos was silenced. Our study identified the transcription factor Fos as a key regulator of ILTV infection through its transcription factor function on both the virus and host sides, improving the current understanding of both avian herpesvirus–host interactions and the roles of AP-1 in viral infection.