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1.
Nat Med ; 25(12): 1873-1884, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31806906

RESUMO

Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-ß renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/ß stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.


Assuntos
Encefalite por Herpes Simples/genética , Herpesvirus Humano 1/genética , Neurônios/imunologia , RNA Nucleolar Pequeno/genética , Adulto , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/virologia , Pré-Escolar , Encefalite por Herpes Simples/imunologia , Encefalite por Herpes Simples/patologia , Encefalite por Herpes Simples/virologia , Feminino , Predisposição Genética para Doença , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/patogenicidade , Humanos , Imunidade/genética , Lactente , Masculino , Metagenoma/genética , Metagenoma/imunologia , Pessoa de Meia-Idade , Neurônios/virologia , RNA Nucleolar Pequeno/imunologia
2.
Indian J Ophthalmol ; 67(7): 1040-1046, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31238404

RESUMO

Purpose: To determine the presence of herpes simplex virus and varicella zoster virus (HSV 1 and 2, VZV) in the cornea of normal subjects by multiplex real time quantitative (qPCR) assay and evaluate its utility in the diagnosis of viral keratitis. Methods: Corneal epithelial cells from 33 eyes of 22 patients undergoing photorefractive keratectomy surgery (controls) and 50 corneal scrapings from 50 patients with suspected HSV keratitis were analyzed for the presence of HSV1 by conventional PCR and for presence of HSV1 and 2 and/or VZV by multiplex real-time PCR. Corneal scrapings of patients were also tested for HSV1 antigen by immunofluorescence assay (IFA). The results were compared and clinical records reviewed. Results: HSV1 and VZV DNA were detected in 8/33 controls (mean-14.3 ± 7.96, range: 3-29.1 copies/mL) and 2/33 controls (mean-10.7 ± 10.9, range 3-18.5 copies/ml) respectively. HSV2 was not detected in any of the controls. Copy numbers above the mean + 1SD of controls were considered significant for viral load in patient samples. Significantly higher number of corneal scrapings (39/50, 78%) from patients were positive for HSV1 (1.2 × 106 copies/mL ± 3.7 × 106 copies/mL) by real time qPCR compared to IFA (11/48, 23%, P value 0.0001) and conventional PCR (20/50, 40%, P value 0.0002). Double infection with HSV-1 (1.5 × 107 copies/ml) and HSV-2 (3.57 × 104 copies/ml) in one case and VZV infection (1.03 × 102 copies/ml) in another was also detected by the multiplex real-time PCR. Conclusion: Multiplex real-time PCR reliably detects HSV1 and 2 and VZV DNA and is ideal for the diagnosis of HSV and VZV keratitis in an ocular microbiology laboratory.


Assuntos
Infecções Oculares Virais/diagnóstico , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Herpesvirus Humano 3/genética , Ceratite Herpética/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecção pelo Vírus da Varicela-Zoster/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Córnea/patologia , Córnea/virologia , DNA Viral/análise , Infecções Oculares Virais/epidemiologia , Infecções Oculares Virais/virologia , Feminino , Seguimentos , Herpes Simples/diagnóstico , Herpes Simples/epidemiologia , Herpes Simples/virologia , Humanos , Índia/epidemiologia , Lactente , Ceratite Herpética/epidemiologia , Ceratite Herpética/virologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Infecção pelo Vírus da Varicela-Zoster/epidemiologia , Infecção pelo Vírus da Varicela-Zoster/virologia , Adulto Jovem
3.
Cornea ; 38(9): 1185-1188, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31205162

RESUMO

PURPOSE: To report a case of severe bilateral necrotizing herpes simplex keratitis (HSK) in an immunocompetent patient, with genotyping of the underlying herpes simplex virus 1 (HSV-1). METHODS: Genetic analyses of HSV-1 in tear samples were performed with polymerase chain reaction-based restriction fragment length polymorphism, targeting the viral genes unique short (US)2, US4 (glycoprotein G), and US7 (glycoprotein I). RESULTS: A 64-year-old woman with no history of atopy or immune disorders manifested bilateral keratitis with geographic ulcer. Her initial visual acuity was 20/1000 (OD) and 20/20 (OS). Polymerase chain reaction testing of a tear sample revealed the presence of HSV-1 in both eyes, and the patient was diagnosed with bilateral HSK. Both eyes progressed to necrotizing keratitis during the treatment course. Continuous intensive treatment, at first with acyclovir ointment and oral valacyclovir and later with steroid eye drops for stromal keratitis, finally improved the patient's condition. However, after 2 years, her visual acuity was limited to 20/250 (OD) and 20/60 (OS) because of corneal opacity from scarring. We found that the strain in the current case had a genotype combination of C/A/B (for US2/US4/US7), a known pattern in Japan, in both eyes. CONCLUSIONS: We successfully performed an unprecedented genetic analysis of an HSV-1 strain isolated from a case of bilateral necrotizing HSK in an immunocompetent patient. The association of the HSV-1 genotype with the clinical manifestation remains unclear, calling for more data from new cases, especially from different geographic regions.


Assuntos
Herpesvirus Humano 1/genética , Ceratite Herpética/virologia , DNA Viral/análise , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição
4.
BMJ Case Rep ; 12(5)2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31138593

RESUMO

A 60-year-old man with a history of severe herpes simplex virus type 1 (HSV-1) encephalitis 2 years prior presented with acute onset of visual loss in the left eye. Dilated funduscopic examination showed retinitis and occlusive vasculitis with retinal necrosis. PCR of the vitreous fluid was positive for HSV-1, and he was diagnosed with acute retinal necrosis (ARN) due to HSV-1. The patient was treated with intravenous acyclovir and intravitreous foscarnet for 2 weeks, followed by high dose oral valacyclovir for 2 weeks. He was subsequently placed on planned life-long suppressive valacyclovir. His case demonstrates that acute visual loss concomitant with or subsequent to HSV-1 encephalitis warrants suspicion of ARN. Prompt therapy with effective antiviral medication is necessary to reduce the risk of sight-threatening complications. Chronic suppression with oral antiviral therapy after ARN is recommended to prevent involvement of the contralateral eye, though there is no consensus on the duration and dosage of antivirals.


Assuntos
Encefalite por Herpes Simples/complicações , Encefalite por Herpes Simples/diagnóstico , Síndrome de Necrose Retiniana Aguda/diagnóstico por imagem , Síndrome de Necrose Retiniana Aguda/etiologia , Doença Aguda , Aciclovir/administração & dosagem , Aciclovir/uso terapêutico , Antivirais/uso terapêutico , Diagnóstico Diferencial , Encefalite por Herpes Simples/virologia , Infecções Oculares Virais/complicações , Infecções Oculares Virais/diagnóstico , Foscarnet/administração & dosagem , Foscarnet/uso terapêutico , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/isolamento & purificação , Humanos , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Oftalmoscópios , Doenças Raras , Síndrome de Necrose Retiniana Aguda/tratamento farmacológico , Síndrome de Necrose Retiniana Aguda/virologia , Resultado do Tratamento , Valaciclovir/administração & dosagem , Valaciclovir/uso terapêutico
5.
Nature ; 570(7760): 257-261, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31142842

RESUMO

Herpesviruses are enveloped viruses that are prevalent in the human population and are responsible for diverse pathologies, including cold sores, birth defects and cancers. They are characterized by a highly pressurized pseudo-icosahedral capsid-with triangulation number (T) equal to 16-encapsidating a tightly packed double-stranded DNA (dsDNA) genome1-3. A key process in the herpesvirus life cycle involves the recruitment of an ATP-driven terminase to a unique portal vertex to recognize, package and cleave concatemeric dsDNA, ultimately giving rise to a pressurized, genome-containing virion4,5. Although this process has been studied in dsDNA phages6-9-with which herpesviruses bear some similarities-a lack of high-resolution in situ structures of genome-packaging machinery has prevented the elucidation of how these multi-step reactions, which require close coordination among multiple actors, occur in an integrated environment. To better define the structural basis of genome packaging and organization in herpes simplex virus type 1 (HSV-1), we developed sequential localized classification and symmetry relaxation methods to process cryo-electron microscopy (cryo-EM) images of HSV-1 virions, which enabled us to decouple and reconstruct hetero-symmetric and asymmetric elements within the pseudo-icosahedral capsid. Here we present in situ structures of the unique portal vertex, genomic termini and ordered dsDNA coils in the capsid spooled around a disordered dsDNA core. We identify tentacle-like helices and a globular complex capping the portal vertex that is not observed in phages, indicative of herpesvirus-specific adaptations in the DNA-packaging process. Finally, our atomic models of portal vertex elements reveal how the fivefold-related capsid accommodates symmetry mismatch imparted by the dodecameric portal-a longstanding mystery in icosahedral viruses-and inform possible DNA-sequence recognition and headful-sensing pathways involved in genome packaging. This work showcases how to resolve symmetry-mismatched elements in a large eukaryotic virus and provides insights into the mechanisms of herpesvirus genome packaging.


Assuntos
Microscopia Crioeletrônica , Empacotamento do DNA , Genoma Viral , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/ultraestrutura , Conformação de Ácido Nucleico , Capsídeo/química , Capsídeo/ultraestrutura , DNA Viral/química , DNA Viral/ultraestrutura , Herpesvirus Humano 1/química , Modelos Moleculares , Vírion/química , Vírion/genética , Vírion/ultraestrutura
6.
BMC Genomics ; 20(1): 382, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31096907

RESUMO

BACKGROUND: Several lines of evidence suggest that recombination plays a central role in replication and evolution of herpes simplex virus-1 (HSV-1). G-quadruplex (G4)-motifs have been linked to recombination events in human and microbial genomes, but their role in recombination has not been studied in DNA viruses. RESULTS: The availability of near full-length sequences from 40 HSV-1 recombinant strains with exact position of the recombination breakpoints provided us with a unique opportunity to investigate the role of G4-motifs in recombination among herpes viruses. We mapped the G4-motifs in the parental and all the 40 recombinant strains. Interestingly, the genome-wide distribution of breakpoints closely mirrors the G4 densities in the HSV-1 genome; regions of the genome with higher G4 densities had higher number of recombination breakpoints. Biophysical characterization of oligonucleotides from a subset of predicted G4-motifs confirmed the formation of G-quadruplex structures. Our analysis also reveals that G4-motifs are enriched in regions flanking the recombination breakpoints. Interestingly, about 11% of breakpoints lie within a G4-motif, making these DNA secondary structures hotspots for recombination in the HSV-1 genome. Breakpoints within G4-motifs predominantly lie within G4-clusters rather than individual G4-motifs. Of note, we identified the terminal guanosine of G4-clusters at the boundaries of the UL (unique long) region on either side of the OriL (origin of replication within UL) represented the commonest breakpoint among the HSV-1 recombinants. CONCLUSION: Our findings suggest a correlation between the HSV-1 recombination landscape and the distribution of G4-motifs and G4-clusters, with possible implications for the evolution of DNA viruses.


Assuntos
Pontos de Quebra do Cromossomo , DNA Viral/genética , Quadruplex G , Genoma Viral , Herpesvirus Humano 1/genética , Recombinação Genética , Replicação do DNA , Humanos
7.
Virol Sin ; 34(4): 386-396, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31020575

RESUMO

To date, 29 distinct microRNAs (miRNAs) have been reported to be expressed during herpes simplex virus infections. Sequence analysis of mature herpes simplex virus-1 (HSV-1) miRNAs revealed five sets of miRNAs that are complementary to each other: miR-H6-5p/H1-3p, miR-H6-3p/H1-5p, H2-5p/H14-3p, miR-H2-3p/H14-5p, and miR-H7/H27. However, the roles of individual miRNAs and consequences of this complementarity remain unclear. Here, we focus on two of these complementary miRNAs, miR-H6-5p and miR-H1-3p, using loss-of-function experiments in vitro and in a mouse model of infection using an miRNA sponge approach, including tandem multiplex artificial miRNA-binding sequences that do not match perfectly to the target miRNA inserted downstream of a green fluorescent protein reporter gene. Infection with recombinant virus expressing the miR-H6-5p sponge reduced viral protein levels and virus yield. Decreased accumulation of viral proteins was also observed at early stages of infection in the presence of both an miR-H6-5p inhibitor and plasmid-expressed miR-H1-3p. Moreover, establishment of latency and reactivation did not differ between the recombinant virus expressing the miR-H6-5p sponge and wild-type HSV-1. Taken together, these data suggest that miR-H6-5p has an as-yet-unidentified role in the early stages of viral infection, and its complement miR-H1-3p suppresses this role in later stages of infection. This report extends understanding of the roles of miRNAs in infection by herpes simplex viruses, supporting a model of infection in which the production of virus and its virulent effects are tightly controlled to maximize persistence in the host and population.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/genética , MicroRNAs/genética , Proteínas Virais/genética , Replicação Viral , Animais , Linhagem Celular , Herpesvirus Humano 1/fisiologia , Mutação com Perda de Função , Camundongos , RNA Viral/genética , Latência Viral
8.
J Biol Chem ; 294(21): 8412-8423, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-30944173

RESUMO

The innate immune system plays an essential role in initial recognition of pathogen infection by producing inflammatory cytokines and type I interferons. cGAS is a cytoplasmic sensor for DNA derived from DNA viruses. cGAS binding with DNA induces the production of cGAMP, a second messenger that associates with STING in endoplasmic reticulum (ER). STING changes its cellular distribution from ER to perinuclear Golgi, where it activates the protein kinase TBK1 that catalyzes the phosphorylation of IRF3. Here we found that STING trafficking is regulated by myotubularin-related protein (MTMR) 3 and MTMR4, members of protein tyrosine phosphatases that dephosphorylate 3' position in phosphatidylinositol (PtdIns) and generate PtdIns5P from PtdIns3,5P2 and PtdIns from PtdIns3P. We established MTMR3 and MTMR4 double knockout (DKO) RAW264.7 macrophage cells and found that they exhibited increased type I interferon production after interferon-stimulatory DNA (ISD) stimulation and herpes simplex virus 1 infection concomitant with enhanced IRF3 phosphorylation. In DKO cells, STING rapidly trafficked from ER to Golgi after ISD stimulation. Notably, DKO cells exhibited enlarged cytosolic puncta positive for PtdIns3P and STING was aberrantly accumulated in this puncta. Taken together, these results suggest that MTMR3 and MTMR4 regulate the production of PtdIns3P, which plays a critical role in suppressing DNA-mediated innate immune responses via modulating STING trafficking.


Assuntos
DNA Viral/imunologia , Herpesvirus Humano 1/imunologia , Imunidade Inata , Macrófagos/imunologia , Proteínas de Membrana/imunologia , Fosfatos de Fosfatidilinositol/imunologia , Proteínas Tirosina Fosfatases não Receptoras/imunologia , Animais , DNA Viral/genética , Herpesvirus Humano 1/genética , Proteínas de Membrana/genética , Camundongos , Fosfatos de Fosfatidilinositol/genética , Transporte Proteico/genética , Transporte Proteico/imunologia , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Células RAW 264.7
9.
J Med Microbiol ; 68(5): 748-754, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30938666

RESUMO

PURPOSE: Herpes simplex virus (HSV) is a common lifelong sexually transmitted infection. HSV-1 typically manifests as oral cold sores, while HSV-2 is more traditionally associated with sexual transmission and infection. We have developed a real-time PCR (Trioplex) for the simultaneous detection of HSV-1 and -2 and the bacterial phage internal control (IC) MS2. METHODOLOGY: To determine the performance of the Trioplex method and resolve discrepancies, 178 clinical specimens from cutaneous and mucocutaneous sources were tested using 3 different methods; virus culture with direct fluorescent antibody (DFA) immunostaining, Trioplex and a commercially available HSV analyte-specific reagent (ASR). RESULTS: HSV Trioplex was significantly more sensitive than virus culture (89 and 67 % HSV 1/2, respectively) and comparable to the commercial assay (P<0.001). Cost analysis revealed that the Trioplex reduced cost by 80  % compared to cell culture. CONCLUSIONS: The implementation of the HSV Trioplex improved the detection turnaround time from 3-10 days to 2.5 h, thus streamlining Herpes detection, improving sensitivity and reducing laboratory costs.


Assuntos
Herpes Simples/diagnóstico , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Reação em Cadeia da Polimerase em Tempo Real/economia , Pele/virologia , Custos e Análise de Custo , DNA Viral/análise , Feminino , Técnica Direta de Fluorescência para Anticorpo , Herpes Genital/diagnóstico , Herpes Genital/virologia , Herpes Simples/virologia , Herpesvirus Humano 1/isolamento & purificação , Herpesvirus Humano 2/isolamento & purificação , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Pele/patologia
10.
Mol Cell ; 74(3): 466-480.e4, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30930055

RESUMO

The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2ß-DNA cleavage complex (TOP2ßcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2ßcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.


Assuntos
DNA Topoisomerases Tipo II/genética , Herpesvirus Humano 1/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-akt/genética , Latência Viral/genética , Animais , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Herpesvirus Humano 1/patogenicidade , Humanos , Proteína Homóloga a MRE11/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Neurônios/metabolismo , Neurônios/virologia , Fosforilação , Ratos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética
11.
Braz J Microbiol ; 50(3): 663-668, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31001794

RESUMO

Polymorphisms in the structural gene MBL-2 (mannose-binding lectin-2) may result in low MBL serum concentration, associated with greater susceptibility to infection. The study evaluated the effects of MBL-2 polymorphisms with the oral manifestations of the HSV in human immunodeficiency virus (HIV)-infected patients. An observational case-control study was carried out, with the sample comprising 64 HIV+ and 65 healthy individuals. The signs and symptoms of HSV oral infection were evaluated, and oral mucosa buccal smears were collected. Polymorphisms of the MBL-2 gene and HSV-1 DNA were amplified through real-time PCR. The data revealed that of 64 HIV+, 29.6% presented signs and symptoms of HSV oral infection. Of these, the HSV-1 DNA was detected through real-time PCR in 21% of cases, and in 13.3% of asymptomatic individuals. There was no statistically significant difference between the symptomatic (p = 1) and the asymptomatic (p = 0.52) individuals, HIV+ and HIV-. Different genotypes (AA, A0, or 00) did not contribute to the oral manifestation of HSV in the HIV+ patients (p = 0.81) or HIV- (p = 0.45). There was no statistically significant difference in either group (p = 0.52). No significant association was identified between the MBL-2 gene polymorphisms in the oral manifestation of HSV infection. However, further studies are recommended with larger population groups before discarding this interrelationship.


Assuntos
Infecções por HIV/complicações , Herpes Simples/genética , Herpesvirus Humano 1/fisiologia , Lectina de Ligação a Manose/genética , Boca/virologia , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Genótipo , Infecções por HIV/genética , HIV-1/genética , HIV-1/isolamento & purificação , HIV-1/fisiologia , Herpes Simples/etiologia , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
12.
Retrovirology ; 16(1): 9, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940160

RESUMO

BACKGROUND: We previously showed that the gM of HSV-1 could restrict the release of infectious HIV-1 from cells. In this study, we analyzed if the four HSV-1 glycoproteins (gD, gB, and gH/gL), which are the minimum glycoproteins required for HSV-1 entry, restricted the release of infectious HIV-1. RESULTS: Of these four glycoproteins, gD and gH/gL restricted the production of infectious HIV-1 from cells transfected with an infectious molecular clone of HIV-1 (strain NL4-3) while gB had no significant effect. Pulse-chase analyses indicated that gD did not affect the biosynthesis and processing of gp160 into gp120/gp41, the transport of the gp120/gp41 to the cell surface, or the release of HIV-1 particles from the cell surface. Our analyses revealed that gD was incorporated into HIV-1 virus particles while gp120/gp41 was excluded from released virus particles. Truncated mutants of gD revealed that the cytoplasmic domain was dispensable but that a membrane bound gD was required for the restriction of release of infectious HIV-1. Finally, cell lines expressing gD also potently restricted the release of infectious virus. CONCLUSIONS: Due to its ability to exclude HIV-1 gp120/gp41 from maturing virus, gD may provide a useful tool in deciphering mechanisms of Env incorporation into maturing virus particles.


Assuntos
HIV-1/fisiologia , Herpesvirus Humano 1/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Linhagem Celular , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/metabolismo , Herpesvirus Humano 1/genética , Humanos , Glicoproteínas de Membrana/genética , Proteínas do Envelope Viral/genética
13.
mSphere ; 4(1)2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814317

RESUMO

More than 14,000 neonates are infected with herpes simplex virus (HSV) annually. Approximately half display manifestations limited to the skin, eyes, or mouth (SEM disease). The rest develop invasive infections that spread to the central nervous system (CNS disease or encephalitis) or throughout the infected neonate (disseminated disease). Invasive HSV disease is associated with significant morbidity and mortality, but the viral and host factors that predispose neonates to these forms are unknown. To define viral diversity within the infected neonatal population, we evaluated 10 HSV-2 isolates from newborns with a range of clinical presentations. To assess viral fitness independently of host immune factors, we measured viral growth characteristics in cultured cells and found diverse in vitro phenotypes. Isolates from neonates with CNS disease were associated with larger plaque size and enhanced spread, with the isolates from cerebrospinal fluid (CSF) exhibiting the most robust growth. We sequenced complete viral genomes of all 10 neonatal viruses, providing new insights into HSV-2 genomic diversity in this clinical setting. We found extensive interhost and intrahost genomic diversity throughout the viral genome, including amino acid differences in more than 90% of the viral proteome. The genes encoding glycoprotein G (gG; US4), glycoprotein I (gI; US7), and glycoprotein K (gK; UL53) and viral proteins UL8, UL20, UL24, and US2 contained variants that were found in association with CNS isolates. Many of these viral proteins are known to contribute to cell spread and neurovirulence in mouse models of CNS disease. This report represents the first application of comparative pathogen genomics to neonatal HSV disease.IMPORTANCE Herpes simplex virus (HSV) causes invasive disease in half of infected neonates, resulting in significant mortality and permanent cognitive morbidity. The factors that contribute to invasive disease are not understood. This study revealed diversity among HSV isolates from infected neonates and detected the first associations between viral genetic variations and clinical disease manifestations. We found that viruses isolated from newborns with encephalitis showed enhanced spread in culture. These viruses contained protein-coding variations not found in viruses causing noninvasive disease. Many of these variations were found in proteins known to impact neurovirulence and viral spread between cells. This work advances our understanding of HSV diversity in the neonatal population and how it may impact disease outcome.


Assuntos
Variação Genética , Herpes Simples/virologia , Herpesvirus Humano 2/genética , Complicações Infecciosas na Gravidez/virologia , Linhagem Celular , Encefalite Viral/virologia , Feminino , Genoma Viral , Genômica , Genótipo , Idade Gestacional , Herpes Simples/complicações , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/isolamento & purificação , Herpesvirus Humano 2/patogenicidade , Humanos , Recém-Nascido , Masculino , Fenótipo , Gravidez , Proteínas Virais/genética
14.
BMJ Case Rep ; 12(3)2019 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-30852508

RESUMO

A young lactating woman presenting to us with simultaneous bilateral corneal lesions was clinically diagnosed to have herpes simplex keratitis, which was confirmed by herpes simplex virus (HSV) PCR. The patient was administered topical and systemic acyclovir therapy and therapeutic penetrating keratoplasty was done in right eye. She was advised to continue breast feeding under strict hygienic conditions. Diagnosis and management of HSV keratitis in a lactating patient can be particularly challenging for both clinician and patient and adoption of a multidisciplinary approach is necessary to ensure safety of mother and child. At 3 months follow-up, the baby was clinically healthy, there were no side effects of acyclovir therapy in the mother or the baby and the patient showed no evidence of recurrence in either eye.


Assuntos
Aciclovir/uso terapêutico , Ceratite Herpética/diagnóstico , Ceratoplastia Penetrante/métodos , Lactação/efeitos dos fármacos , Aciclovir/administração & dosagem , Assistência ao Convalescente , Antivirais/uso terapêutico , Aleitamento Materno , Doenças da Córnea/diagnóstico , Doenças da Córnea/virologia , Diagnóstico Diferencial , Feminino , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/virologia , Herpesvirus Humano 1/genética , Humanos , Ceratite Herpética/tratamento farmacológico , Ceratite Herpética/terapia , Ceratite Herpética/virologia , Doenças Raras , Resultado do Tratamento , Adulto Jovem
15.
J Virol ; 93(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30760568

RESUMO

A majority of adults in Finland are seropositive carriers of herpes simplex viruses (HSV). Infection occurs at epithelial or mucosal surfaces, after which virions enter innervating nerve endings, eventually establishing lifelong infection in neurons of the sensory or autonomic nervous system. Recent data have highlighted the genetic diversity of HSV-1 strains and demonstrated apparent geographic patterns in strain similarity. Though multiple HSV-1 genomes have been sequenced from Europe to date, there is a lack of sequenced genomes from the Nordic countries. Finland's history includes at least two major waves of human migration, suggesting the potential for diverse viruses to persist in the population. Here, we used HSV-1 clinical isolates from Finland to test the relationship between viral phylogeny, genetic variation, and phenotypic characteristics. We found that Finnish HSV-1 isolates separated into two distinct phylogenetic groups, potentially reflecting historical waves of human (and viral) migration into Finland. Each HSV-1 isolate harbored a distinct set of phenotypes in cell culture, including differences in the amount of virus production, extracellular virus release, and cell-type-specific fitness. Importantly, the phylogenetic clusters were not predictive of any detectable pattern in phenotypic differences, demonstrating that whole-genome relatedness is not a proxy for overall viral phenotype. Instead, we highlight specific gene-level differences that may contribute to observed phenotypic differences, and we note that strains from different phylogenetic groups can contain the same genetic variations.IMPORTANCE Herpes simplex viruses (HSV) infect a majority of adults. Recent data have highlighted the genetic diversity of HSV-1 strains and demonstrated apparent genomic relatedness between strains from the same geographic regions. We used HSV-1 clinical isolates from Finland to test the relationship between viral genomic and geographic relationships, differences in specific genes, and characteristics of viral infection. We found that viral isolates from Finland separated into two distinct groups of genomic and geographic relatedness, potentially reflecting historical patterns of human and viral migration into Finland. These Finnish HSV-1 isolates had distinct infection characteristics in multiple cell types tested, which were specific to each isolate and did not group according to genomic and geographic relatedness. This demonstrates that HSV-1 strain differences in specific characteristics of infection are set by a combination of host cell type and specific viral gene-level differences.


Assuntos
Variação Genética , Genoma Viral , Herpes Simples/genética , Herpesvirus Humano 1/genética , Filogenia , Animais , Feminino , Finlândia , Herpesvirus Humano 1/isolamento & purificação , Humanos , Masculino , Células Vero , Sequenciamento Completo do Genoma
16.
Front Immunol ; 10: 2, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30723467

RESUMO

Robust anti-tumor immunity requires innate as well as adaptive immune responses. We have shown that plasmacytoid dendritic cells develop killer cell-like activity in melanoma cell cocultures after exposure to the infectious but replication-deficient herpes simplex virus 1 (HSV-1) d106S. To combine this innate effect with an enhanced adaptive immune response, the gene encoding human MelanA/MART-1 was inserted into HSV-1 d106S via homologous recombination to increase direct expression of this tumor antigen. Infection of Vero cells using this recombinant virus confirmed MelanA expression by Western blotting, flow cytometry, and immunofluorescence. HSV-1 d106S-MelanA induced expression of the transgene in fibroblast and melanoma cell lines not naturally expressing MelanA. Infection of a melanoma cell line with CRISPR-Cas9-mediated knockout of MelanA confirmed de novo expression of the transgene in the viral context. Dependent on MelanA expression, infected fibroblast and melanoma cell lines induced degranulation of HLA-matched MelanA-specific CD8+ T cells, followed by killing of infected cells. To study infection of immune cells, we exposed peripheral blood mononuclear cells and in vitro-differentiated macrophages to the parental HSV-1 d106S, resulting in expression of the transgene GFP in CD11c+ cells and macrophages. These data provide evidence that the application of MelanA-encoding HSV-1 d106S could enhance adaptive immune responses and re-direct MelanA-specific CD8+ T cells to tumor lesions, which have escaped adaptive immune responses via downregulation of their tumor antigen. Hence, HSV-1 d106S-MelanA harbors the potential to induce innate immune responses in conjunction with adaptive anti-tumor responses by CD8+ T cells, which should be evaluated in further studies.


Assuntos
Expressão Gênica , Vetores Genéticos/genética , Herpesvirus Humano 1 , Antígeno MART-1/genética , Vírus Oncolíticos , Transgenes , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Marcação de Genes , Engenharia Genética , Herpesvirus Humano 1/genética , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Melanoma , Vírus Oncolíticos/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo
17.
Methods Mol Biol ; 1937: 177-188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30706396

RESUMO

Herpes simplex virus (HSV) is one of the most extensively studied oncolytic virus platforms. The recent FDA approval of talimogene laherparepvec (T-VEC) has been accelerating translational research of oncolytic HSV (oHSV) as a promising therapeutic for refractory cancers such as glioblastoma, the deadliest primary malignancy in the brain. The large genome size of HSV readily allows arming of oHSV by incorporating therapeutic transgenes within the virus, as exemplified by T-VEC carrying GM-CSF, thereby enhancing the anticancer activity of oHSV. Here we describe a bacterial artificial chromosome-based method for construction of an oHSV expressing a transgene, which we routinely use in the laboratory to create a number of different recombinant oHSV bearing either therapeutic or reporter genes.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Herpesvirus Humano 1/genética , Vírus Oncolíticos/genética , Animais , Vetores Genéticos/genética , Humanos , Terapia Viral Oncolítica , Transfecção , Transgenes , Células Vero
18.
Toxins (Basel) ; 11(2)2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30791373

RESUMO

A set of herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the light chains (LC) of botulinum neurotoxins (BoNT) A, B, C, D, E and F was constructed. Their properties have been assessed in primary cultures of rat embryonic dorsal root ganglia (DRG) neurons, and in organotypic cultures of explanted DRG from adult rats. Following infection of primary cultures of rat embryonic DRG neurons, the different BoNT LC induced efficient cleavage of their corresponding target Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) protein (VAMP, SNAP25, syntaxin). A similar effect was observed following infection by BoNT-A LC of organotypic cultures of adult rat DRG. To quantify and compare the functional activities of the different BoNT LC, the inhibition of calcitonin gene-related protein (CGRP) secretion was assessed in DRG neurons following infection by the different vectors. All BoNT-LC were able to inhibit CGRP secretion although to different levels. Vectors expressing BoNT-F LC displayed the highest inhibitory activity, while those expressing BoNT-D and -E LC induced a significantly lower CGRP release inhibition. Cleavage of SNARE proteins and inhibition of CGRP release could be detected in neuron cultures infected at less than one transducing unit (TU) per neuron, showing the extreme efficacy of these vectors. To our knowledge this is the first study investigating the impact of vector-expressed transgenic BoNT LC in sensory neurons.


Assuntos
Toxinas Botulínicas/genética , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Gânglios Espinais/metabolismo , Herpesvirus Humano 1/genética , Neurotoxinas/genética , Proteínas SNARE/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Células Cultivadas , Feminino , Gânglios Espinais/virologia , Vetores Genéticos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/virologia
19.
Nat Commun ; 10(1): 754, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30765700

RESUMO

Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns. Direct RNA sequencing (direct RNA-seq) using nanopore arrays offers an exciting alternative whereby individual polyadenylated RNAs are sequenced directly, without the recoding and amplification biases inherent to other sequencing methodologies. Here we use direct RNA-seq to profile the herpes simplex virus type 1 (HSV-1) transcriptome during productive infection of primary cells. We show how direct RNA-seq data can be used to define transcription initiation and RNA cleavage sites associated with all polyadenylated viral RNAs and demonstrate that low level read-through transcription produces a novel class of chimeric HSV-1 transcripts, including a functional mRNA encoding a fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L. Thus, direct RNA-seq offers a powerful method to characterize the changing transcriptional landscape of viruses with complex genomes.


Assuntos
Genes Virais/genética , Herpesvirus Humano 1/genética , Nanoporos , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Linhagem Celular , Células Cultivadas , Células Epiteliais/virologia , Fibroblastos/virologia , Genoma Viral/genética , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Neurônios/citologia , Neurônios/virologia , RNA Viral/genética , Proteínas Virais/genética
20.
Clin Immunol ; 200: 19-23, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30639657

RESUMO

Conflicting results have been reported regarding human herpes virus (HHV) reactivation in patients with rheumatoid arthritis (RA). To explore this link, 74 RA patients were selected and compared to 42 first degree relatives (FDR) from probands with RA and 25 healthy controls from the Tatarstan women cohort. The serological analysis was done by testing anti-HSV/CMV/EBV IgM, IgG, plus the IgG avidity index, and completed by evaluating HSV/CMV/EBV DNA by PCR. Results from these analyses reveal: (i) a long lasting infection of HHV in RA, FDR and healthy controls (IgG seroconversion >97%); (ii) an elevated IgM anti-HHV response in seroconverted RA patients which is related to HSV1/2 reactivation (HSV1/2 PCR+); and (iii) a multi-reactive IgM HHV burden profile associated with disease activity (DAS28). In conclusion, HSV1/2 reactivation in seroconverted RA patients is associated with an abnormal anti-HHV immune response, which was reflected in IgM HHV burden, and in activity disease profile.


Assuntos
Anticorpos Antivirais/imunologia , Artrite Reumatoide/imunologia , Herpesviridae/imunologia , Imunoglobulina M/imunologia , Ativação Viral/imunologia , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/virologia , Estudos de Casos e Controles , Citomegalovirus/genética , Citomegalovirus/imunologia , DNA Viral/análise , Família , Feminino , Herpesviridae/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/imunologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Soroconversão , Índice de Gravidade de Doença
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