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Nature Communications May 2024Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a...
Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.
Topics: Animals; Gene Editing; Female; Dependovirus; Mice; Herpesvirus 1, Human; Herpes Simplex; Viral Load; Virus Shedding; Disease Models, Animal; Virus Latency; Humans; Genetic Vectors; Vero Cells; Genetic Therapy; Herpes Genitalis; DNA, Viral
PubMed: 38740820
DOI: 10.1038/s41467-024-47940-y -
Epidemiology and Infection May 2024Nosocomial outbreak of varicella zoster virus (VZV) has been reported when susceptible individuals encounter a case of chicken pox or shingles. A suspected VZV outbreak...
Nosocomial outbreak of varicella zoster virus (VZV) has been reported when susceptible individuals encounter a case of chicken pox or shingles. A suspected VZV outbreak was investigated in a 50-bedded in-patient facility of Physical Medicine and Rehabilitation in a tertiary care multispecialty hospital. A 30-year-old female patient admitted with Pott's spine was clinically diagnosed with chicken pox on 31 December 2022. The following week, four more cases were identified in the same ward. All cases were diagnosed as laboratory-confirmed varicella zoster infection by PCR. Primary case was a housekeeping staff who was clinically diagnosed with chicken pox 3 weeks prior (9 December 2022). He returned to work on eighth day of infection (17 December 2022) after apparent clinical recovery but before the lesions had crusted over. Thirty-one HCWs were identified as contacts a and three had no evidence of immunity. Two of these susceptible HCWs had onset of chickenpox shortly after first dose of VZV vaccination was inoculated. All cases recovered after treatment with no reported complications. VZV infection is highly contagious in healthcare settings with susceptible populations. Prompt identification of cases and implementation of infection prevention and control measures like patient isolation and vaccination are essential for the containment of outbreaks.
Topics: Humans; Disease Outbreaks; Tertiary Care Centers; Adult; Female; India; Cross Infection; Herpesvirus 3, Human; Male; Chickenpox; Long-Term Care; Varicella Zoster Virus Infection
PubMed: 38736415
DOI: 10.1017/S0950268824000712 -
International Journal of Molecular... May 2024Acquiring resistance against antiviral drugs is a significant problem in antimicrobial therapy. In order to identify novel antiviral compounds, the antiviral activity of...
Acquiring resistance against antiviral drugs is a significant problem in antimicrobial therapy. In order to identify novel antiviral compounds, the antiviral activity of eight plants indigenous to the southern region of Hungary against herpes simplex virus-2 (HSV-2) was investigated. The plant extracts and the plant compound carnosic acid were tested for their effectiveness on both the extracellular and intracellular forms of HSV-2 on Vero and HeLa cells. HSV-2 replication was measured by a direct quantitative PCR (qPCR). Among the tested plant extracts, () exhibited a 90.46% reduction in HSV-2 replication at the 0.47 μg/mL concentration. Carnosic acid, a major antimicrobial compound found in rosemary, also demonstrated a significant dose-dependent inhibition of both extracellular and intracellular forms of HSV-2. The 90% inhibitory concentration (IC) of carnosic acid was between 25 and 6.25 μg/mL. Proteomics and high-resolution respirometry showed that carnosic acid suppressed key ATP synthesis pathways such as glycolysis, citrate cycle, and oxidative phosphorylation. Inhibition of oxidative phosphorylation also suppressed HSV-2 replication up to 39.94-fold. These results indicate that the antiviral action of carnosic acid includes the inhibition of ATP generation by suppressing key energy production pathways. Carnosic acid holds promise as a potential novel antiviral agent against HSV-2.
Topics: Abietanes; Virus Replication; Chlorocebus aethiops; Vero Cells; Adenosine Triphosphate; Humans; Animals; Herpesvirus 2, Human; Antiviral Agents; Plant Extracts; HeLa Cells
PubMed: 38732202
DOI: 10.3390/ijms25094983 -
International Journal of Molecular... May 2024Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and...
Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threatening encephalitis. At present, there are no vaccines available, and some existing antiviral treatments can be ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In this report, the in vitro anti-HSV effect of 9,9'-norharmane dimer (nHo-dimer), which belongs to the β-carboline (βC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and did not impede either the attachment or penetration steps of viral particles. The antiviral effect was only exerted under the constant presence of the dimer in the incubation media, and the mechanism of action was found to involve later events of virus infection. Analysis of fluorescence lifetime imaging data showed that the nHo-dimer internalized well into the cells when present in the extracellular incubation medium, with a preferential accumulation into perinuclear organelles including mitochondria. After washing the host cells with fresh medium free of nHo-dimer, the signal decreased, suggesting the partial release of the compound from the cells. This agrees with the observation that the antiviral effect is solely manifested when the alkaloid is consistently present in the incubation media.
Topics: Antiviral Agents; Chlorocebus aethiops; Humans; Vero Cells; Animals; Simplexvirus; Herpes Simplex; Carbolines; Herpesvirus 1, Human; Harmine
PubMed: 38732185
DOI: 10.3390/ijms25094966 -
International Journal of Molecular... Apr 2024Although Herpes simplex virus type 1 (HSV-1) has been deeply studied, significant gaps remain in the fundamental understanding of HSV-host interactions: our work focused...
Although Herpes simplex virus type 1 (HSV-1) has been deeply studied, significant gaps remain in the fundamental understanding of HSV-host interactions: our work focused on studying the Infected Cell Protein 27 (ICP27) as an inhibitor of the Absent-in-melanoma-2 (AIM 2) inflammasome pathway, leading to reduced pro-inflammatory cytokines that influence the activation of a protective innate immune response to infection. To assess the inhibition of the inflammasome by the ICP27, hTert-immortalized Retinal Pigment Epithelial cells (hTert-RPE 1) infected with HSV-1 wild type were compared to HSV-1 lacking functional ICP27 (HSV-1∆ICP27) infected cells. The activation of the inflammasome by HSV-1∆ICP27 was demonstrated by quantifying the gene and protein expression of the inflammasome constituents using real-time PCR and Western blot. The detection of the cleavage of the pro-caspase-1 into the active form was performed by using a bioluminescent assay, while the quantification of interleukins 1β (IL-1β) and 18 (IL-18)released in the supernatant was quantified using an ELISA assay. The data showed that the presence of the ICP27 expressed by HSV-1 induces, in contrast to HSV-1∆ICP27 vector, a significant downregulation of AIM 2 inflammasome constituent proteins and, consequently, the release of pro-inflammatory interleukins into the extracellular environment reducing an effective response in counteracting infection.
Topics: Humans; Inflammasomes; Herpesvirus 1, Human; Cytokines; Immediate-Early Proteins; Retinal Pigment Epithelium; Epithelial Cells; Cell Line; Herpes Simplex; DNA-Binding Proteins
PubMed: 38731826
DOI: 10.3390/ijms25094608 -
Molecules (Basel, Switzerland) Apr 2024Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a promising nucleic acid-based gene targeting approach for gene expression knock-down...
Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a promising nucleic acid-based gene targeting approach for gene expression knock-down and modulation. The RNase P-EGS strategy is unique as an EGS can be designed to basepair any mRNA sequence and recruit intracellular RNase P for hydrolysis of the target mRNA. In this study, we provide the first direct evidence that the RNase P-based approach effectively blocks the gene expression and replication of herpes simplex virus 2 (HSV-2), the causative agent of genital herpes. We constructed EGSs to target the mRNA encoding HSV-2 single-stranded DNA binding protein ICP8, which is essential for viral DNA genome replication and growth. In HSV-2 infected cells expressing a functional EGS, ICP8 levels were reduced by 85%, and viral growth decreased by 3000 folds. On the contrary, ICP8 expression and viral growth exhibited no substantial differences between cells expressing no EGS and those expressing a disabled EGS with mutations precluding RNase P recognition. The anti-ICP8 EGS is specific in targeting ICP8 because it only affects ICP8 expression but does not affect the expression of the other viral immediate-early and early genes examined. This study shows the effective and specific anti-HSV-2 activity of the RNase P-EGS approach and demonstrates the potential of EGS RNAs for anti-HSV-2 applications.
Topics: Herpesvirus 2, Human; Virus Replication; Gene Expression Regulation, Viral; Humans; Ribonuclease P; Animals; Viral Proteins; Chlorocebus aethiops; RNA, Messenger; Vero Cells; Immediate-Early Proteins; DNA-Binding Proteins
PubMed: 38731543
DOI: 10.3390/molecules29092052 -
International Journal of Oral Science May 2024N-methyladenosine (mA) RNA methylation is critical for regulating mRNA translation; however, its role in the development, progression, and immunotherapy response of head...
N-methyladenosine (mA) RNA methylation is critical for regulating mRNA translation; however, its role in the development, progression, and immunotherapy response of head and neck squamous cell carcinoma (HNSCC) remains largely unknown. Using Tgfbr1 and Pten conditional knockout (2cKO) mice, we found the neoplastic transformation of oral mucosa was accompanied by increased mA modification levels. Analysis of mA-associated genes identified TRMT61A as a key mA writer linked to cancer progression and poor prognosis. Mechanistically, TRMT61A-mediated tRNA-mA modification promotes MYC protein synthesis, upregulating programmed death-ligand 1 (PD-L1) expression. Moreover, mA modification levels were also elevated in tumors treated with oncolytic herpes simplex virus (oHSV), contributing to reactive PD-L1 upregulation. Therapeutic mA inhibition sustained oHSV-induced antitumor immunity and reduced tumor growth, representing a promising strategy to alleviate resistance. These findings indicate that mA inhibition can prevent immune escape after oHSV therapy by reducing PD-L1 expression, providing a mutually reinforcing combination immunotherapy approach.
Topics: Animals; B7-H1 Antigen; Mice; Proto-Oncogene Proteins c-myc; Oncolytic Viruses; Signal Transduction; Humans; Adenosine; Down-Regulation; Squamous Cell Carcinoma of Head and Neck; Oncolytic Virotherapy; PTEN Phosphohydrolase; Mice, Knockout; Head and Neck Neoplasms; Simplexvirus; Cell Line, Tumor
PubMed: 38730256
DOI: 10.1038/s41368-024-00304-0 -
Nature Communications May 2024Encephalitis is a rare and potentially fatal manifestation of herpes simplex type 1 infection. Following genome-wide genetic analyses, we identified a previously...
Encephalitis is a rare and potentially fatal manifestation of herpes simplex type 1 infection. Following genome-wide genetic analyses, we identified a previously uncharacterized and very rare heterozygous variant in the E3 ubiquitin ligase WWP2, in a 14-month-old girl with herpes simplex encephalitis. The p.R841H variant (NM_007014.4:c.2522G > A) impaired TLR3 mediated signaling in inducible pluripotent stem cells-derived neural precursor cells and neurons; cells bearing this mutation were also more susceptible to HSV-1 infection compared to control cells. The p.R841H variant increased TRIF ubiquitination in vitro. Antiviral immunity was rescued following the correction of p.R841H by CRISPR-Cas9 technology. Moreover, the introduction of p.R841H in wild type cells reduced such immunity, suggesting that this mutation is linked to the observed phenotypes.
Topics: Humans; Ubiquitin-Protein Ligases; Female; Encephalitis, Herpes Simplex; Infant; Mutation; Herpesvirus 1, Human; Induced Pluripotent Stem Cells; Toll-Like Receptor 3; Ubiquitination; Neurons; Neural Stem Cells; CRISPR-Cas Systems
PubMed: 38730242
DOI: 10.1038/s41467-024-48287-0 -
PloS One 2024Marek's disease (MD) is an important neoplastic disease caused by serotype 1 Marek's disease virus (MDV-1), which results in severe economic losses worldwide. Despite...
Marek's disease (MD) is an important neoplastic disease caused by serotype 1 Marek's disease virus (MDV-1), which results in severe economic losses worldwide. Despite vaccination practices that have controlled the MD epidemic, current increasing MD-suspected cases indicate the persistent viral infections circulating among vaccinated chicken farms in many countries. However, the lack of available information about phylogeny and molecular characterization of circulating MDV-1 field strains in Taiwan reveals a potential risk in MD outbreaks. This study investigated the genetic characteristics of 18 MDV-1 strains obtained from 17 vaccinated chicken flocks in Taiwan between 2018 and 2020. Based on the sequences of the meq oncogene, the phylogenetic analysis demonstrated that the circulating Taiwanese MDV-1 field strains were predominantly in a single cluster that showed high similarity with strains from countries of the East Asian region. Because the strains were obtained from CVI988/Rispens vaccinated chicken flocks and the molecular characteristics of the Meq oncoprotein showed features like vvMDV and vv+MDV strains, the circulating Taiwanese MDV-1 field strains may have higher virulence compared with vvMDV pathotype. In conclusion, the data presented demonstrates the circulation of hypervirulent MDV-1 strains in Taiwan and highlights the importance of routine surveillance and precaution strategies in response to the emergence of enhanced virulent MDV-1.
Topics: Animals; Chickens; Herpesvirus 2, Gallid; Marek Disease; Marek Disease Vaccines; Oncogene Proteins, Viral; Phylogeny; Poultry Diseases; Taiwan; Vaccination; Virulence
PubMed: 38728352
DOI: 10.1371/journal.pone.0303371 -
The Veterinary Quarterly Dec 2024Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe...
Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 10 TCID/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.
Topics: Animals; Vaccines, Attenuated; Ducks; Poultry Diseases; Fibroblasts; Chick Embryo; Viral Vaccines; Mardivirus; Herpesviridae Infections; India
PubMed: 38726839
DOI: 10.1080/01652176.2024.2350668