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Purpose: To sequence, identify, and perform phylogenetic and recombination analysis on three clinical adenovirus samples taken from the vitreous humor at the Bascom Palmer Eye Institute., Methods: The PacBio Sequel II was used to sequence the genomes of the three clinical adenovirus isolates. To identify the isolates, a full genome-based multiple sequence alignment (MSA) of 722 mastadenoviruses was generated using multiple alignment using fast Fourier transform (MAFFT). MAFFT was also used to generate genome-based human adenovirus B (HAdV-B) MSAs, as well as HAdV-B fiber, hexon, and penton protein-based MSAs. To examine recombination within HAdV-B, RF-Net 2 and Bootscan software programs were used., Results: In the course of classifying three new atypical ocular adenovirus samples, taken from the vitreous humor, we found that all three isolates were HAdV-B species. The three Bascom Palmer HAdV-B genomes were then combined with over 300 HAdV-B genome sequences, including nine ocular HAdV-B genome sequences. Attempts to categorize the penton, hexon, and fiber serotypes using phylogeny of the three Bascom Palmer samples were inconclusive due to incongruence between serotype and phylogeny in the dataset. Recombination analysis using a subset of HAdV-B strains to generate a hybridization network detected recombination between nonhuman primate and human-derived strains, recombination between one HAdV-B strain and the HAdV-E outgroup, and limited recombination between the B1 and B2 clades., Conclusions: The discordance between serotype and phylogeny detected in this study suggests that the current classification system does not accurately describe the natural history and phylogenetic relationships among adenoviruses.

The retinoic acid-inducible gene I (RIG)-I-like receptor (RLR) family of RNA sensor proteins plays a key role in the innate immune response to viral nucleic acids, including viral gene delivery vectors, but little is known about the expression of RLR proteins in the retina. The purpose of this study was to characterize cell-specific expression patterns of RLR proteins in non-human primate (NHP) neural retina tissue and to examine if RLR pathway signaling restricts viral gene delivery transduction. Since RLR protein signaling converges at the mitochondrial antiviral signaling protein (MAVS), experiments were performed to determine if knockdown of MAVS affected FIVGFP transduction efficiency in the human Mueller cell line MIO-M1. Immunoblotting confirmed expression of RIG-I, melanoma differentiation-associated protein 5 (MDA5), laboratory of genetics and physiology 2 (LGP2), and MAVS proteins in MIO-M1 cells and NHP retina tissue. Double label immunofluorescence (IF) studies revealed RIG-I, LGP2, and MAVS were expressed in Mueller microglial cells in the NHP retina. In addition, LGP2 and MDA5 proteins were detected in cone and retinal ganglion cells (RGC). MDA5 was also present in a subset of calretinin positive amacrine cells, and in nuclei within the inner nuclear layer (INL). Knockdown of MAVS significantly increased the transduction efficiency of the lentiviral vector FIVGFP in MIO-M1 cells, compared to control cells. FIVGFP or AAVGFP challenge did not alter expression of the LGP2, MAVS, MDA5 or RIG-I genes in MIO-M1 cells or NHP retina tissue compared to media treated controls. Our data demonstrate that innate immune response proteins involved in viral RNA sensing, including MDA5, RIG-I, LGP2, and MAVS, are expressed in several cell types within the NHP neural retina. In addition, the MAVS protein restricts non-human lentiviral transduction efficiency in MIO-M1 cells., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: There are limited data on the prevalence and genetic diversity of herpes simplex virus type 1 (HSV-1) virulence genes in ocular isolates. Here, we sequenced 36 HSV-1 ocular isolates, collected by the Bascom Palmer Eye Institute, a university-based eye hospital, from three different ocular anatomical sites (conjunctiva, cornea, and eyelid) and carried out a genomic and phylogenetic analyses., Methods: The PacBio Sequel II long read platform was used for genome sequencing. Phylogenetic analysis and genomic analysis were performed to help better understand genetic variability among common virulence genes in ocular herpetic disease., Results: A phylogenetic network generated using the genome sequences of the 36 Bascom Palmer ocular isolates, plus 174 additional strains showed that ocular isolates do not group together phylogenetically. Analysis of the thymidine kinase and DNA polymerase protein sequences from the Bascom Palmer isolates showed multiple novel single nucleotide polymorphisms, but only one, BP-K14 encoded a known thymidine kinase acyclovir resistance mutation. An analysis of the multiple sequence alignment comprising the 51 total ocular isolates versus 159 nonocular strains detected several possible single nucleotide polymorphisms in HSV-1 genes that were found significantly more often in the ocular isolates. These genes included UL6, gM, VP19c, VHS, gC, VP11/12, and gG., Conclusions: There does not seem to be a specific genetic feature of viruses causing ocular infection. The identification of novel and common recurrent polymorphisms may help to understand the drivers of herpetic pathogenicity and specific factors that may influence the virulence of ocular disease.

Sex related differences in the incidence or severity of infection have been described for multiple viruses. With herpes simplex viruses, the best example is HSV-2 genital infection where women have a higher incidence of infection and can have more severe infections than men. HSV-1 causes several types of infections including skin and mucosal ulcers, keratitis, and encephalitis in humans that do not appear to have a strong biological sex component. Given that mouse strains differ in their MHC loci it is important to determine if sex differences occur in multiple strains of mice. Our goal was to answer two questions: Are virus related sex differences present in BALB/C mice and does virulence of the viral strain have an effect? We generated a panel of recombinant HSV-1 viruses with differing virulence phenotypes and characterized multiple clinical correlates of ocular infection in BALB/c mice. We found no sex-specific differences in blepharitis, corneal clouding, neurovirulence, and viral titers in eye washes. Sex differences in neovascularization, weight loss and eyewash titers were observed for some recombinants, but these were not consistent across the phenotypes tested for any recombinant virus. Considering these findings, we conclude that there are no significant sex specific ocular pathologies in the parameters measured, regardless of the virulence phenotype following ocular infection in BALB/c mice, suggesting that the use of both sexes is not necessary for the bulk of ocular infection studies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kolb et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Members of the family Herpesviridae have enveloped, spherical virions with characteristic complex structures consisting of symmetrical and non-symmetrical components. The linear, double-stranded DNA genomes of 125-241 kbp contain 70-170 genes, of which 43 have been inherited from an ancestral herpesvirus. In general, herpesviruses have coevolved with and are highly adapted to their hosts, which comprise many mammalian, avian and reptilian species. Following primary infection, they are able to establish lifelong latent infection, during which there is limited viral gene expression. Severe disease is usually observed only in the foetus, the very young, the immunocompromised or following infection of an alternative host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Herpesviridae , which is available at ictv.global/report/herpesviridae.

The goal of this study was to determine the expression and distribution of the host restriction factors (RFs) TRIM5α and TRIM11 in non-human primate (NHP) neural retina tissue and the human Muller cell line MIO-M1. In addition, experiments were performed to determine the effect of TRIM5α and TRIM11 knockdown on FIVGFP transduction of MIO-M1 cells with the goal of devising strategies to increase the efficiency of lentiviral (LV) gene delivery. Immunofluorescence (IF) studies indicated that TRIM5α and TRIM11 were localized predominantly in nuclei within the outer nuclear layer (ONL) and inner nuclear layer (INL) of NHP retina tissue. Double label IF indicated that TRIM5α and TRIM11 were localized to some of the retinal Muller cell nuclei. MIO-M1 cells expressed TRIM5α predominantly in the nucleus and TRIM11 primarily in the cytosol. FIVGFP transduction efficiency was significantly increased, at 4 and 7 days post transduction, in TRIM5α and TRIM11 knockdown clones (KD) compared to WT MIO-M1 cells. In addition, pretreatment with the proteasome inhibitor MG132 increased the transduction efficiency of FIVGFP in WT MIO-M1 cells. The nuclear translocation of NF-κB (p65), at 72 h post FIVGFP transduction, was enhanced in TRIM5α and TRIM11 KD clones. The expression of TRIM5α and TRIM11 in macaque neural retina tissue and MIO-M1 cells indicate the presence of these RFs in NHP retina and human Muller cells. Our data indicate that even partial knockdown of TRIM5α or TRIM11, or a short proteasome inhibitor pretreatment, can increase the transduction efficiency of a LV vector., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Herpes simplex viruses form a genus within the alphaherpesvirus subfamily, with three identified viral species isolated from Old World monkeys (OWM); Macacine alphaherpesvirus 1 (McHV-1; herpes B), Cercopithecine alphaherpesvirus 2 (SA8), and Papiine alphaherpesvirus 2 (PaHV-2; herpes papio). Herpes B is endemic to macaques, while PaHV-2 and SA8 appear endemic to baboons. All three viruses are genetically and antigenically similar, with SA8 and PaHV-2 thought to be avirulent in humans, while herpes B is a biosafety level 4 pathogen. Recently, next-generation sequencing (NGS) has resulted in an increased number of published OWM herpes simplex genomes, allowing an encompassing phylogenetic analysis., Results: In this study, phylogenetic networks, in conjunction with a genome-based genetic distance cutoff method were used to examine 27 OWM monkey herpes simplex isolates. Genome-based genetic distances were calculated, resulting in distances between lion and pig-tailed simplex viruses themselves, and versus herpes B core strains that were higher than those between PaHV-2 and SA8 (approximately 14 and 10% respectively). The species distance cutoff was determined to be 8.94%, with the method recovering separate species status for PaHV-2 and SA8 and showed that lion and pig-tailed simplex viruses (vs core herpes B strains) were well over the distance species cutoff., Conclusions: We propose designating lion and pig-tailed simplex viruses as separate, individual viral species, and that this may be the first identification of viral cryptic species.

Selenium is an essential micronutrient for living beings, as it helps to maintain the normal physiological functions of the organism. The numerous discoveries involving the importance of this element to the health of human beings have fostered interest in research to develop enriched and functional foods. The present study evaluated the potential for bacterial strains of Enterococcus faecalis (CH121 and CH124), Lactobacillus parabuchneri (ML4), Lactobacillus paracasei (ML13, ML33, CH135, and CH139), and Lactobacillus plantarum (CH131) to bioaccumulate Se in their biomass by adding different concentrations of sodium selenite (30 to 200 mg/L) to the culture medium. Quantification of Se with UV and visible molecular absorption spectroscopy showed that the investigated bacteria were able to bioaccumulate this micromineral into their biomass. Two of the L. paracasei strains (ML13 and CH135) bioaccumulated the highest Se concentrations (38.1 ± 1.7 mg/g and 40.7 ± 1.1 mg/g, respectively) after culture in the presence of 150 mg/L of Se. This bioaccumulation potential has applications in the development of dairy products and may be an alternative Se source in the diets of humans and other animals., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The purpose of this study was to characterize cell-specific expression patterns of Toll-like receptors (TLR) in non-human primate (NHP) neural retina tissue. TLR 4, 5, 6, and 7 proteins were detected by immunblotting of macaque retina tissue lysates and quantitative PCR (qPCR) demonstrated TLRs 4-7 mRNA expression. Immunofluorescence (IF) microscopy detected TLRs 4-7 in multiple cell types in macaque neural retina including Muller, retinal ganglion cells (RGC), amacrine, and bipolar cells. These results demonstrate that TLRs 4-7 are constitutively expressed by neurons in the NHP retina raising the possibility that these cells could be involved in retinal innate inflammatory responses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Feline herpes virus type 1 (FHV-1) is widely considered to be the leading cause of ocular disease in cats and has been implicated in upper respiratory tract infections. Little, however is known about interstrain phylogenetic relationships, and details of the genomic structure. For the present study, twenty-six FHV-1 isolates from different cats in animal shelters were collected from eight separate locations in the USA, and the genomes sequenced. Genomic characterization of these isolates includied short sequence repeat (SSR) detection, with fewer SSRs detected, compared to herpes simplex viruses type 1 and 2. For phylogenetic and recombination analysis, 27 previously sequenced isolates of FHV-1 were combined with the 26 strains sequenced for the present study. The overall genomic interstrain genetic distance between all available isolates was 0.093%. Phylogenetic analysis identified four main FHV-1 clades primarily corresponding to geographical collection site. Recombination analysis suggested that interclade recombination has occurred., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: To determine if proteasome inhibition using MG132 increased the efficiency of FIV vector-mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS)., Methods: TM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 μM MG132 and transduced with FIV.GFP (green fluorescent protein)- or FIV.mCherry-expressing vector at a multiplicity of transduction (MOT) of 20. At 24 hours, cells were fixed and stained with antibodies for GFP, and positive cells were counted, manually or by fluorescence-activated cell sorting (FACS). Cells transduced with FIV.GFP particles alone were used as controls. The effect of 20 μM MG132 treatment on high- and low-dose (2 × 107 and 0.8 × 107 transducing units [TU], respectively) FIV.GFP transduction with or without MG132 was also evaluated in MOCAS using fluorescence microscopy. Vector genome equivalents in cells and tissues were quantified by quantitative (q)PCR on DNA., Results: In the MG132 treatment groups, there was a significant dose-dependent increase in the percentage of transduced cells at all concentrations tested. Vector genome equivalents were also increased in TM-1 cells treated with MG132. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20 μM MG132 and the high dose of vector. Vector genome equivalents were also significantly increased in the MOCAS tissues. Increased transduction was not seen with the low dose of virus., Conclusions: Proteasome inhibition increased the transduction efficiency of FIV particles in TM-1 cells and MOCAS and may be a useful adjunct for delivery of therapeutic genes to the TM by lentiviral vectors.

Background: The varicelloviruses comprise a genus within the alphaherpesvirus subfamily, and infect both humans and other mammals. Recently, next-generation sequencing has been used to generate genomic sequences of several members of the Varicellovirus genus. Here, currently available varicellovirus genomic sequences were used for phylogenetic, recombination, and genetic distance analysis., Results: A phylogenetic network including genomic sequences of individual species, was generated and suggested a potential restriction between the ungulate and non-ungulate viruses. Intraspecies genetic distances were higher in the ungulate viruses (pseudorabies virus (SuHV-1) 1.65%, bovine herpes virus type 1 (BHV-1) 0.81%, equine herpes virus type 1 (EHV-1) 0.79%, equine herpes virus type 4 (EHV-4) 0.16%) than non-ungulate viruses (feline herpes virus type 1 (FHV-1) 0.0089%, canine herpes virus type 1 (CHV-1) 0.005%, varicella-zoster virus (VZV) 0.136%). The G + C content of the ungulate viruses was also higher (SuHV-1 73.6%, BHV-1 72.6%, EHV-1 56.6%, EHV-4 50.5%) compared to the non-ungulate viruses (FHV-1 45.8%, CHV-1 31.6%, VZV 45.8%), which suggests a possible link between G + C content and intraspecies genetic diversity. Varicellovirus clade nomenclature is variable across different species, and we propose a standardization based on genomic genetic distance. A recent study reported no recombination between sequenced FHV-1 strains, however in the present study, both splitstree, bootscan, and PHI analysis indicated recombination. We also found that the recently sequenced Brazilian CHV-1 strain BTU-1 may contain a genetic signal in the UL50 gene from an unknown varicellovirus., Conclusion: Together, the data contribute to a greater understanding of varicellovirus genomics, and we also suggest a new clade nomenclature scheme based on genetic distances.

Staphylococcus aureus infection of the cornea is a significant threat to vision. The percentage of bacterial isolates resistant to antibiotics is increasing as is the percentage of infections caused by methicillin resistant isolates. There is a critical need for additional therapeutic approaches and their development will require the use of animal models to test efficacy. Two mouse models of S. aureus keratitis have been described but only quantified stromal keratitis (corneal clouding and perforation). We have extended these models using the methicillin resistant S. aureus USA300 LAC strain and show that eyelid inflammation and swelling (blepharitis) and corneal neovascularization can be quantified. This expanded model should prove useful in assessing additional effects of antibacterial therapies and additional pathological mechanisms involved in bacterial ocular infection.

Unlabelled: Herpes simplex virus 1 (HSV-1) most commonly causes recrudescent labial ulcers; however, it is also the leading cause of infectious blindness in developed countries. Previous research in animal models has demonstrated that the severity of HSV-1 ocular disease is influenced by three main factors: host innate immunity, host immune response, and viral strain. We have previously shown that mixed infection with two avirulent HSV-1 strains (OD4 and CJ994) results in recombinants with a wide range of ocular disease phenotype severity. Recently, we developed a quantitative trait locus (QTL)-based computational approach (vQTLmap) to identify viral single nucleotide polymorphisms (SNPs) predicted to influence the severity of the ocular disease phenotypes. We have now applied vQTLmap to identify HSV-1 SNPs associated with corneal neovascularization and mean peak percentage weight loss (MPWL) using 65 HSV-1 OD4-CJ994 recombinants. The vQTLmap analysis using Random Forest for neovascularization identified phenotypically meaningful nonsynonymous SNPs in the ICP4, UL41 (VHS), UL42, UL46 (VP11/12), UL47 (VP13/14), UL48 (VP22), US3, US4 (gG), US6 (gD), and US7 (gI) coding regions. The ICP4 gene was previously identified as a corneal neovascularization determinant, validating the vQTLmap method. Further analysis detected an epistatic interaction for neovascularization between a segment of the unique long (UL) region and a segment of the inverted repeat short (IRS)/unique short (US) region. Ridge regression was used to identify MPWL-associated nonsynonymous SNPs in the UL1 (gL), UL2, UL4, UL49 (VP22), UL50, and ICP4 coding regions. The data provide additional insights into virulence gene and epistatic interaction discovery in HSV-1., Importance: Herpes simplex virus 1 (HSV-1) typically causes recurrent cold sores; however, it is also the leading source of infectious blindness in developed countries. Corneal neovascularization is critical for the progression of blinding ocular disease, and weight loss is a measure of infection severity. Previous HSV-1 animal virulence studies have shown that the severity of ocular disease is partially due to the viral strain. In the current study, we used a recently described computational quantitative trait locus (QTL) approach in conjunction with 65 HSV-1 recombinants to identify viral single nucleotide polymorphisms (SNPs) involved in neovascularization and weight loss. Neovascularization SNPs were identified in the ICP4, VHS, UL42, VP11/12, VP13/14, VP22, gG, US3, gD, and gI genes. Further analysis revealed an epistatic interaction between the UL and US regions. MPWL-associated SNPs were detected in the UL1 (gL), UL2, UL4, VP22, UL50, and ICP4 genes. This approach will facilitate future HSV virulence studies., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Injection of herpes simplex virus vectors into the vitreous of primate eyes induces an acute, transient uveitis. The purpose of this study was to characterize innate immune responses of macaque neural retina tissue to the herpes simplex virus type 1-based gene delivery vector hrR3. PCR array analysis demonstrated the induction of the pro-inflammatory cytokine IL-6, as well as the anti-inflammatory cytokine IL-10, following hrR3 exposure. Secretion of IL-6 was detected by ELISA and cone photoreceptors and Muller cells were the predominant IL-6 positive cell types. RNA in situ hybridization confirmed that IL-6 was expressed in photoreceptor and Muller cells. The IL-10 positive cells in the inner nuclear layer were identified as amacrine cells by immunofluorescence staining with calretinin antibody. hrR3 challenge resulted in activation of NFκB (p65) in Muller glial cells, but not in cone photoreceptors, suggesting a novel regulatory mechanism for IL-6 expression in cone cells. hrR3 replication was not required for IL-6 induction or NFκB (p65) activation. These data suggest a pro-inflammatory (IL-6)/anti-inflammatory (IL-10) axis exists in neural retina and the severity of acute posterior uveitis may be determined by this interaction. Further studies are needed to identify the trigger for IL-6 and IL-10 induction and the mechanism of IL-6 induction in cone cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unlabelled: Vaccinia virus (VACV) keratitis is a serious complication following smallpox vaccination and can lead to blindness. The pathological mechanisms involved in ocular VACV infection are poorly understood. Previous studies have used rabbits, but the lack of immune reagents and transgenic or knockout animals makes them less suitable for mechanistic studies. We report that infection of C57BL/6 mice with 1 × 10(7) PFU of vaccinia virus strain WR results in blepharitis, corneal neovascularization, and stromal keratitis. The DryVax strain of VACV was completely attenuated. Infection required corneal scarification and replication-competent virus, and the severity of ocular disease was similar in 4- to 6-week-old and 1-year-old mice. Viral titers peaked at approximately 1 × 10(6) PFU on day 5 postinfection, and virus had not cleared by day 13 postinfection. Neutrophils were found in the peripheral cornea on day 1 after infection and then declined, followed by infiltration of both CD4(+) and CD8(+) T cells, which remained peripheral throughout the infection. Blood vessel growth extended 2 to 5 mm into the cornea from the limbus. Infection of CD4(-/-), CD8(-/-), or antibody-depleted mice resulted in similar disease severity and corneal clouding, indicating that both T-cell subsets were involved in the immunopathological response. Depletion of both CD4(+) and CD8(+) T cells resulted in significantly more severe disease and failure to clear the virus. On the basis of our results, the pathology of VACV keratitis is significantly different from that of herpes simplex virus keratitis. Further studies are likely to reveal novel information regarding virulence and immune responses to viral ocular infection., Importance: Potentially blinding eye infections can occur after vaccination for smallpox. Very little is known about the pathological mechanisms that are involved, and the information that is available was generated using rabbit models. The lack of immunological reagents for rabbits makes such studies difficult. We characterized a mouse model of vaccinia virus ocular disease using C57BL/6 mice and strain WR and show that both CD4(+) and CD8(+) T-cell subsets play a role in the blinding eye disease and in controlling virus replication. On the basis of these results, vaccinia virus keratitis is significantly different from herpes simplex virus keratitis, and further studies using this model should generate novel insights into immunopathological responses to viral ocular infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Herpes simplex virus type 1 causes mucocutaneous lesions, and is the leading cause of infectious blindness in the United States. Animal studies have shown that the severity of HSV-1 ocular disease is influenced by three main factors; innate immunity, host immune response and viral strain. We previously showed that mixed infection with two avirulent HSV-1 strains (OD4 and CJ994) resulted in recombinants that exhibit a range of disease phenotypes from severe to avirulent, suggesting epistatic interactions were involved. The goal of this study was to develop a quantitative trait locus (QTL) analysis of HSV-1 ocular virulence determinants and to identify virulence associated SNPs. Blepharitis and stromal keratitis quantitative scores were characterized for 40 OD4:CJ994 recombinants. Viral titers in the eye were also measured. Virulence quantitative trait locus mapping (vQTLmap) was performed using the Lasso, Random Forest, and Ridge regression methods to identify significant phenotypically meaningful regions for each ocular disease parameter. The most predictive Ridge regression model identified several phenotypically meaningful SNPs for blepharitis and stromal keratitis. Notably, phenotypically meaningful nonsynonymous variations were detected in the UL24, UL29 (ICP8), UL41 (VHS), UL53 (gK), UL54 (ICP27), UL56, ICP4, US1 (ICP22), US3 and gG genes. Network analysis revealed that many of these variations were in HSV-1 regulatory networks and viral genes that affect innate immunity. Several genes previously implicated in virulence were identified, validating this approach, while other genes were novel. Several novel polymorphisms were also identified in these genes. This approach provides a framework that will be useful for identifying virulence genes in other pathogenic viruses, as well as epistatic effects that affect HSV-1 ocular virulence.

Cytochrome P450 epoxygenase isozymes convert free arachidonic acid into eicosanoids named epoxyeicosatrienoic acids (EETs) that have roles in regulating inflammation. EETs are rapidly converted to dihydroxyeicosatrienoic acids (DiHETs) by soluble epoxide hydrolase (sEH). Little is known about the potential role of these metabolites in uveitis, but conversion of EETs to DiHETs could contribute to the inflammation. We tested a potent and orally available inhibitor of sEH for its ability to reduce ocular inflammation in a rabbit LPS-induced model of uveitis. Rabbits were treated by subcutaneous injection with the sEH inhibitor (UC1728, 3 mg/kg), or the vehicle control (PEG400) and uveitis was assessed at 6, 24 and 48 h post-intracameral LPS injection using a modified Hackett-McDonald scoring system. Eyes treated by intra-cameral injection of PBS, or by aseptic preparation served as further controls. Signs of inflammation in this model were mild and transient. Treatment with UC1728 did not significantly reduce inflammation compared to animals treated with the PEG400 vehicle. Blood levels of UC1728 were a thousand fold higher than the in vitro determined inhibitory potency (IC50) of the compound suggesting a significant degree of inhibition of sEH in the rabbit. The lack of efficacy suggests that sEH or its substrates the EETs may not be involved in mediating inflammation in this model of uveitis.

Unlabelled: Herpes simplex virus 1 (HSV-1) causes recurrent mucocutaneous ulcers and is the leading cause of infectious blindness and sporadic encephalitis in the United States. HSV-1 has been shown to be highly recombinogenic; however, to date, there has been no genome-wide analysis of recombination. To address this, we generated 40 HSV-1 recombinants derived from two parental strains, OD4 and CJ994. The 40 OD4-CJ994 HSV-1 recombinants were sequenced using the Illumina sequencing system, and recombination breakpoints were determined for each of the recombinants using the Bootscan program. Breakpoints occurring in the terminal inverted repeats were excluded from analysis to prevent double counting, resulting in a total of 272 breakpoints in the data set. By placing windows around the 272 breakpoints followed by Monte Carlo analysis comparing actual data to simulated data, we identified a recombination bias toward both high GC content and intergenic regions. A Monte Carlo analysis also suggested that recombination did not appear to be responsible for the generation of the spontaneous nucleotide mutations detected following sequencing. Additionally, kernel density estimation analysis across the genome found that the large, inverted repeats comprise a recombination hot spot., Importance: Herpes simplex virus 1 (HSV-1) virus is the leading cause of sporadic encephalitis and blinding keratitis in developed countries. HSV-1 has been shown to be highly recombinogenic, and recombination itself appears to be a significant component of genome replication. To date, there has been no genome-wide analysis of recombination. Here we present the findings of the first genome-wide study of recombination performed by generating and sequencing 40 HSV-1 recombinants derived from the OD4 and CJ994 parental strains, followed by bioinformatics analysis. Recombination breakpoints were determined, yielding 272 breakpoints in the full data set. Kernel density analysis determined that the large inverted repeats constitute a recombination hot spot. Additionally, Monte Carlo analyses found biases toward high GC content and intergenic and repetitive regions., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Herpes simplex virus 2 (HSV-2) is a major global pathogen, infecting 16% of people 15 to 49 years old worldwide and causing recurrent genital ulcers. Little is known about viral factors contributing to virulence, and there are currently only two genomic sequences available. In this study, we determined nearly complete genomic sequences of six additional HSV-2 isolates, using Illumina MiSeq. We report that HSV-2 has a genomic overall mean distance of 0.2355%, which is less than that of HSV-1. There were approximately 100 amino-acid-encoding and indels per genome. Microsatellite mapping found a bias toward intergenic regions in the nonconserved microsatellites and a genic bias in all detected tandem repeats. Extensive recombination between the HSV-2 strains was also strongly implied. This was the first study to analyze multiple HSV-2 sequences, and the data will be valuable in future evolutionary, virulence, and structure-function studies., Importance: HSV-2 is a significant worldwide pathogen, causing recurrent genital ulcers. Here we present six nearly complete HSV-2 genomic sequences, and, with the addition of two previously sequenced strains, for the first time genomic, phylogenetic, and recombination analysis was performed on multiple HSV-2 genomes. Our results show that microsatellite mapping found a bias toward intergenic regions in the nonconserved microsatellites and a genic bias in all detected tandem repeats and confirm that chimpanzee herpesvirus 1 (ChHV-1) is a separate species and that each of the HSV-2 strains is a genomic mosaic., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Purpose: Purified Clostridium botulinum exoenzyme C3 transferase (C3) effects on the actin cytoskeleton in human trabecular meshwork cells (HTM) and on the outflow facility response in monkey organ-cultured anterior segments (MOCAS) were determined in the presence or absence of viral vectors., Methods: Human adenovirus type 5 (AdV) and feline immunodeficiency virus (FIV) vectors were produced using kits. Cell soluble purified C3 (C3cs) was purchased commercially. Recombinant C3 (C3rec) cDNA was overexpressed in Escherichia coli and purified. The HTM cells were incubated with up to 10 μg/mL C3cs or with 5 μg of C3rec and/or viral vector (multiplicity of infection [MOI] = 25). Cells then were fixed and stained for actin. Outflow facility in MOCAS was measured at baseline, 4 hours, 24 hours, and 3 to 4 days following bolus injection of AdV (1.6 × 107 transducing units) and/or 2.5 μg C3rec., Results: The HTM cells treated for 4 hours with C3cs (all doses) or for 24 hours with C3rec developed a rounded morphology and lost stress fibers. Cells transduced with vectors alone showed no changes at any time point. Cells exposed to C3rec and cotransduced with either viral vector showed significant disruption of the actin cytoskeleton within 4 hours after exposure, which persisted at 24 hours. In MOCAS, the AdV vector alone had no effect on outflow facility, but enhanced the response to C3rec at 4 hours., Conclusions: Coadministration of viral vectors enhances the ability of C3 transferase to disrupt actin stress fiber formation in HTM cells and increase outflow facility in MOCAS. Viral vectors potentially could be used to increase the bioavailability of proteins for cells that are difficult to transfect.

Microbial pathogens-bacteria, viruses, fungi, and parasites-are significant causes of blindness, particularly in developing countries. For bacterial and some viral infections a number of antimicrobial drugs are available for therapy but there are fewer available for use in treating fungal and parasitic keratitis. There are also problems with current antimicrobials, such as limited efficacy and the presence of drug-resistant microbes. Thus, there is a need to develop additional drugs. Nature has given us an example of 1 potential source of new antimicrobials: antimicrobial peptides and proteins that are either present in bodily fluids and tissues constitutively or are induced upon infection. Given the nature of peptides, topical applications are the most likely use to be successful and this is ideal for treating keratitis. Such peptides would also be active against drug-resistant pathogens and might act synergistically if used in combination therapy. Hundreds of peptides with antimicrobial properties have been isolated or synthesized but only a handful have been tested against ocular pathogens and even fewer have been tested in animal models. This review summarizes the currently available information on the use of peptides to treat keratitis, outlines some of the problems that have been identified, and discusses future studies that will be needed. Most of the peptides that have been tested have shown activity at concentrations that do not warrant further development, but 1 or 2 have promising activity raising the possibility that peptides can be developed to treat keratitis.

Currently, the most effective outflow drugs approved for clinical use are prostaglandin F2α analogues, but these require daily topical self-dosing and have various intraocular, ocular surface and extraocular side effects. Lentiviral vector-mediated delivery of the prostaglandin F synthase (PGFS) gene, resulting in long-term reduction of intraocular pressure (IOP), may eliminate off-target tissue effects and the need for daily topical PGF2α self-administration. Lentiviral vector-mediated delivery of the PGFS gene to the anterior segment has been achieved in cats and non-human primates. Although these results are encouraging, our studies have identified a number of challenges that need to be overcome for prostaglandin gene therapy to be translated into the clinic. Using examples from our work in non-human primates, where we were able to achieve a significant reduction in IOP (2 mm Hg) for 5 months after delivery of the cDNA for bovine PGF synthase, we identify and discuss these issues and consider several possible solutions.

Herpes simplex virus type 1 (HSV-1) is an important human pathogen which requires activation of nuclear factor-kappa B (NFκB) during its replication cycle. The persistent nature of HSV-1 infection, and the emergence of drug-resistant strains, highlights the importance of research to develop new antiviral agents. Toll-like receptors (TLRs) play a prominent role during the early antiviral response by recognizing viral nucleic acid and gene products, activating NFκB, and stimulating the production of inflammatory cytokines. We demonstrate a significant effect on HSV-1 replication in ARPE-19 and Vero cells when oligonucleotides designed to inhibit TLR9 are added 2h prior to infection. A greater than 90% reduction in the yield of infectious virus was achieved at oligonucleotide concentrations of 10-20 μM. TLR9 inhibitory oligonucleotides prevented expression of essential immediate early herpes gene products as determined by immunofluorescence microscopy and Western blotting. TLR9 oligonucleotides also interfered with viral attachment and entry. A TLR9 inhibitory oligonucleotide containing five adjacent guanosine residues (G-ODN) exhibited virucidal activity and inhibited HSV-1 replication when added post-infection. The antiviral effect of the TLR9 inhibitory oligonucleotides did not depend on the presence of TLR9 protein, suggesting a mechanism of inhibition that is not TLR9 specific. TLR9 inhibitory oligonucleotides also reduced NFκB activity in nuclear extracts. Studies using these TLR inhibitors in the context of viral infection should be interpreted with caution., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We compared 31 complete and nearly complete globally derived HSV-1 genomic sequences using HSV-2 HG52 as an outgroup to investigate their phylogenetic relationships and look for evidence of recombination. The sequences were retrieved from NCBI and were then aligned using Clustal W. The generation of a maximum likelihood tree resulted in a six clade structure that corresponded with the timing and routes of past human migration. The East African derived viruses contained the greatest amount of genetic diversity and formed four of the six clades. The East Asian and European/North American derived viruses formed separate clades. HSV-1 strains E07, E22 and E03 were highly divergent and may each represent an individual clade. Possible recombination was analyzed by partitioning the alignment into 5 kb segments, performing individual phylogenetic analysis on each partition and generating a.phylogenetic network from the results. However most evidence for recombination spread at the base of the tree suggesting that recombination did not significantly disrupt the clade structure. Examination of previous estimates of HSV-1 mutation rates in conjunction with the phylogenetic data presented here, suggests that the substitution rate for HSV-1 is approximately 1.38 × 10(-7) subs/site/year. In conclusion, this study expands the previously described HSV-1 three clade phylogenetic structures to a minimum of six and shows that the clade structure also mirrors global human migrations. Given that HSV-1 has co-evolved with its host, sequencing HSV-1 isolated from various populations could serve as a surrogate biomarker to study human population structure and migration patterns.

Agaricus brasiliensis cell-wall polysaccharides isolated from fruiting body (FR) and mycelium (MI) and their respective sulfated derivatives (FR-S and MI-S) were chemically characterized using elemental analysis, TLC, FT-IR, NMR, HPLC, and thermal analysis. Cytotoxic activity was evaluated against A549 tumor cells by MTT and sulforhodamine assays. The average molecular weight (Mw) of FR and MI was estimated to be 609 and 310 kDa, respectively. FR-S (127 kDa) and MI-S (86 kDa) had lower Mw, probably due to hydrolysis occurring during the sulfation reaction. FR-S and MI-S presented ~14% sulfur content in elemental analysis. Sulfation of samples was characterized by the appearance of two new absorption bands at 1253 and 810 cm(-1) in the infrared spectra, related to S=O and C-S-O sulfate groups, respectively. Through (1)H and (13)C NMR analysis FR-S was characterized as a (1→6)-(1→3)-β-D-glucan fully sulfated at C-4 and C-6 terminal and partially sulfated at C-6 of (1→3)-β-D-glucan moiety. MI-S was shown to be a (1→3)-β-D-gluco-(1→2)-β-D-mannan, partially sulfated at C-2, C-3, C-4, and C-6, and fully sulfated at C-6 of the terminal residues. The combination of high degree of sulfation and low molecular weight was correlated with the increased cytotoxic activity (48 h of treatment) of both FR-S (EC₅₀=605.6 μg/mL) and MI-S (EC₅₀=342.1 μg/mL) compared to the non-sulfated polysaccharides FR and MI (EC₅₀>1500 μg/mL)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Agaricus brasiliensis (syn. A. subrufescens), a basidiomycete fungus native to the Atlantic forest in Brazil, contains cell walls rich in glucomannan polysaccharides. The β-(1 → 2)-gluco-β-(1 → 3)-mannan was isolated from A. brasiliensis mycelium, chemically modified by sulfation, and named MI-S. MI-S has multiple mechanisms of action, including inhibition of herpes simplex virus (HSV) attachment, entry, and cell-to-cell spread (F. T. G. S. Cardozo, C. M. Camelini, A. Mascarello, M. J. Rossi, R. J. Nunes, C. R. Barardi, M. M. de Mendonça, and C. M. O. Simões, Antiviral Res. 92:108-114, 2011). The antiherpetic efficacy of MI-S was assessed in murine ocular, cutaneous, and genital infection models of HSV. Groups of 10 mice were infected with HSV-1 (strain KOS) or HSV-2 (strain 333). MI-S was given either topically or by oral gavage under various pre- and posttreatment regimens, and the severity of disease and viral titers in ocular and vaginal samples were determined. No toxicity was observed in the uninfected groups treated with MI-S. The topical and oral treatments with MI-S were not effective in reducing ocular disease. Topical application of MI-S on skin lesions was also not effective, but cutaneously infected mice treated orally with MI-S had significantly reduced disease scores (P < 0.05) after day 9, suggesting that healing was accelerated. Vaginal administration of MI-S 20 min before viral challenge reduced the mean disease scores on days 5 to 9 (P < 0.05), viral titers on day 1 (P < 0.05), and mortality (P < 0.0001) in comparison to the control groups (untreated and vehicle treated). These results show that MI-S may be useful as an oral agent to reduce the severity of HSV cutaneous and mucosal lesions and, more importantly, as a microbicide to block sexual transmission of HSV-2 genital infections.

Purpose: To test the in vivo activity of a peptide derived from the protein transducing domain of the human immunodeficiency virus (HIV) Tat protein, TAT-Cd°, in a murine herpes simplex type 1 (HSV-1) keratitis model., Methods: the efficacy of TAT-CD° was assessed in a postinfection treatment model with different concentrations (1 mg/mL, 0.1 mg/mL, 0.01 mg/mL) of the peptide in one of four delivery vehicles: artificial tears, PBS, methylcellulose, and aquaphor cream. Treatment began within 4 or 24 hours postinfection. Viral titers in the tear film were determined by plaque assay., Results: TAT-Cd° reduced the severity of keratitis in all of the delivery vehicles tested when treatment started, 4 hours postinfection. Peptide in the tears or PBS delivery vehicle had the most significant reduction in disease severity and delayed the onset of vascularization and stromal keratitis. The percentage of mice presenting with disease was also significantly reduced and viral titers were reduced by 1 log at 24 hours postinfection in mice treated with 1 mg/mL TAT-Cd°, suggesting that inhibiting replication early is sufficient to achieve clinical effects. Lower concentrations were not effective and delaying treatment by 24 hours was also not effective., Conclusions: This study shows that TAT-Cd° is an effective antiviral against HSV-1 strain KOS when applied shortly postinfection and that aqueous-based formulations are more suitable.

Herpes simplex ocular infection is a major cause of corneal blindness. Local antiviral treatments exist but are associated with corneal toxicity, and resistance has become an issue. We evaluated the biodistribution and efficacy of a humanized anti-herpes simplex virus (anti-HSV) IgG FAb fragment (AC-8; 53 kDa) following repeated topical administration. AC-8 was found in the corneal epithelium, anterior stroma, subepithelial stromal cells, and retinal glial cells, with preferential entry through the ocular limbus. AC-8 was active against 13 different strains of HSV-1, with 50% and 90% mean effective concentrations (MEC(50) and MEC(90), respectively) ranging from 0.03 to 0.13 μg/ml, indicating broad-spectrum activity. The in vivo efficacy of AC-8 was evaluated in a mouse model of herpes-induced ocular disease. Treatment with low-dose AC-8 (1 mg/ml) slightly reduced the ocular disease scores. A greater reduction of the disease scores was observed in the 10-mg/ml AC-8-treated group, but not as much as with trifluridine (TFT). AC-8 treatment reduced viral titers but less than trifluridine. AC-8 did not display any toxicity to the cornea or other structures in the eye. In summary, topical instillation of an anti-HSV FAb can be used on both intact and ulcerated corneas. It is well tolerated and does not alter reepithelialization. Further studies to improve the antiviral effect are needed for AC-8 to be considered for therapeutic use.

Background: The EB peptide is a 20-mer that was previously shown to have broad spectrum in vitro activity against several unrelated viruses, including highly pathogenic avian influenza, herpes simplex virus type I, and vaccinia, the prototypic orthopoxvirus. To expand on this work, we evaluated EB for in vitro activity against the zoonotic orthopoxviruses cowpox and monkeypox and for in vivo activity in mice against vaccinia and cowpox., Findings: In yield reduction assays, EB had an EC50 of 26.7 μM against cowpox and 4.4 μM against monkeypox. The EC50 for plaque reduction was 26.3 μM against cowpox and 48.6 μM against monkeypox. A scrambled peptide had no inhibitory activity against either virus. EB inhibited cowpox in vitro by disrupting virus entry, as evidenced by a reduction of the release of virus cores into the cytoplasm. Monkeypox was also inhibited in vitro by EB, but at the attachment stage of infection. EB showed protective activity in mice infected intranasally with vaccinia when co-administered with the virus, but had no effect when administered prophylactically one day prior to infection or therapeutically one day post-infection. EB had no in vivo activity against cowpox in mice., Conclusions: While EB did demonstrate some in vivo efficacy against vaccinia in mice, the limited conditions under which it was effective against vaccinia and lack of activity against cowpox suggest EB may be more useful for studying orthopoxvirus entry and attachment in vitro than as a therapeutic against orthopoxviruses in vivo.

Purpose: Little is known about the role of sequence variation in the pathology of HSV-1 keratitis virus. The goal was to show that a multiplex, high-throughput genome-sequencing approach is feasible for simultaneously sequencing seven HSV-1 ocular strains., Methods: A genome sequencer was used to sequence the HSV-1 ocular isolates TFT401, 134, CJ311, CJ360, CJ394, CJ970, and OD4, in a single lane. Reads were mapped to the HSV-1 strain 17 reference genome by high-speed sequencing. ClustalW was used for alignment, and the Mega 4 package was used for phylogenetic analysis (www.megasoftware.net). Simplot was used to compare genetic variability and high-speed sequencing was used to identify SNPs (developed by Stuart Ray, Johns Hopkins University School of Medicine, Baltimore, MD, http://sray.med.som.jhml.edu/SCRoftware/simplot)., Results: Approximately 95% to 99% of the seven genomes were sequenced in a single lane with average coverage ranging from 224 to 1345. Phylogenetic analysis of the sequenced genome regions revealed at least three clades. Each strain had approximately 200 coding SNPs compared to strain 17, and these were evenly spaced along the genomes. Four genes were highly conserved, and six were more variable. Reduced coverage was obtained in the highly GC-rich terminal repeat regions., Conclusions: Multiplex sequencing is a cost-effective way to obtain the genomic sequences of ocular HSV-1 isolates with sufficient coverage of the unique regions for genomic analysis. The number of SNPs and their distribution will be useful for analyzing the genetics of virulence, and the sequence data will be useful for studying HSV-1 evolution and for the design of structure-function studies.

Given the poor immunogenicity of current H5N1 influenza vaccines, additives and adjuvants remain a viable solution for increasing efficacy. Here, we demonstrate that a 20-amino acid peptide (EB) possessing influenza antiviral activity also enhances the immune response to H5N1 vaccination in mice. The addition of EB to formalin-inactivated whole-virus vaccine induced virion aggregation and these aggregates were readily engulfed by phagocytic cells in vitro. In vivo, mice vaccinated with a suboptimal dose of inactivated vaccine containing EB peptide had reduced morbidity, improved viral clearance, and faster recovery than mice receiving vaccine alone. This phenomenon was not accompanied by an increase in virus-specific antibodies. Instead, cell-mediated immunity was enhanced as demonstrated by increased interferon-γ production from splenocytes. This data demonstrates that the EB peptide may a useful adjuvant for boosting the efficacy of poorly immunogenic influenza vaccines., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: The herpes simplex virus type 1 (HSV-1) U(S)1 gene encodes host-range and ocular virulence determinants. Mutations in U(S)1 affecting virulence are known in strain OD4, but the genomic variation across several strains is not known. The goal was to determine the degree of sequence variation in the gene from several ocular HSV isolates., Methods: The U(S)1 gene from six ocular HSV-1 isolates, as well as strains KOS and F, were sequenced, and bioinformatics analyses were applied to the data., Results: Strains 17, F, CJ394, and CJ311 had identical amino acid sequences. With the other strains, most of the variability was concentrated in the amino-terminal third of the protein. MEME analysis identified a 63-residue core sequence (motif 1) present in all α-herpesvirus U(S)1 homologs that were located in a region identified as structured. Ten amino acids were absolutely conserved in all the α-herpesvirus U(S)1 homologs and were all located in the central core. Consensus-binding motifs for cyclin-dependent kinases and pocket proteins were also identified., Conclusions: These results suggest that significant sequence variation exists in the U(S)1 gene, that the α22 protein contains a conserved central core region with structurally variable regions at the amino- and carboxyl termini, that 10 amino acids are conserved in α-herpes U(S)1 homologs, and that additional host proteins may interact with the HSV-1 U(S)1 and U(S)1.5 proteins. This information will be valuable in designing further studies on structure-function relationships and on the role these play in host-range determination and keratitis.

The antiviral peptide, entry blocker (EB), inhibits influenza virus replication by preventing attachment to cells. Here, we identified the minimal and optimal EB sequence that retained antiviral activity with a 50% inhibitory concentration (IC(50)) and 50% effective concentration (EC(50)) similar to those of the full-length EB peptide and several truncated variants that possessed up to 10-fold lower IC(50)s. These data have implications for improving the antiviral efficacy of EB-derived peptides while decreasing production costs and easing synthesis.

Background: Vaccinia virus keratitis (VACVK) is a complication of smallpox vaccination that can result in blindness. There are no Food and Drug Administration-approved treatments for VACVK, and vaccinia immunoglobulin (VIG) is contraindicated in isolated VACVK. We used a rabbit model of infection to compare several therapeutic options for VACVK., Methods: Rabbit eyes were infected with 10(5) plaque-forming units of the Dryvax strain of vaccinia virus and scored daily for 28 days using a modified MacDonald-Shadduck scoring system. Animals were treated for 10 days after the onset of keratitis with albumin, VIG, prednisolone acetate, trifluridine, or combinations thereof. Ocular viral titers and vaccinia-specific antibody titers were determined by plaque assay and enzyme-linked immunosorbent assay, respectively., Results: Treatment with intravenous VIG neither exacerbated nor ameliorated VACVK. Topical prednisolone acetate interfered with viral clearance, and ocular disease rebounded in prednisolone-treated groups. The most effective treatment was topical trifluridine alone., Conclusions: We conclude that (1) VIG did not negatively affect the treatment of isolated keratitis, (2) topical corticosteroids should not be used for treating VACVK, and (3) treatment with topical trifluridine, with or without intravenous VIG, is the preferred therapeutic regimen for treating VACVK.

Unlabelled: Abstract Purpose: To determine if a peptide, TAT-Cd(0), inhibits Herpes simplex virus type 1 infection of human corneal epithelial cells., Methods: TAT-Cd(0) and a control peptide, E(50,51)TAT-Cd(0), were added at various times throughout infection with the lacz-expressing hrR3 virus, and viral replication was measured by β-galactosidase activity. Toxicity was assessed using a dye reduction assay., Results: The CC(50) value for TAT-Cd(0) was ∼100 μM. In assays with peptide present at all times, TAT-Cd(0) was 150-fold more active than E(50,51)TAT-Cd(0) (EC(50) 0.2 vs. 30.0 μM). The EC(50) values of TAT-Cd(0) for entry inhibition, cell protection, virus inactivation, and inhibition of attachment were 0.1, 0.4, 9.5, and 3.0 μM, respectively. TAT-Cd(0) was less effective when added 1 h postinfection (EC(50) = 30.0 μM)., Conclusions: TAT-Cd(0) is an effective inhibitor of Herpes simplex virus type 1 infection in human corneal epithelial cells and affects multiple steps before, or very early, in infection. The peptide has potential as an antiviral and further studies are warranted.

The linear cationic amphiphilic EB peptide, derived from the FGF4 signal sequence, was previously shown to be virucidal and to block herpes simplex type I (HSV-1) entry (H. Bultmann, J. S. Busse, and C. R. Brandt, J. Virol. 75:2634-2645, 2001). Here we show that cells treated with EB (RRKKAAVALLPAVLLALLAP) for less than 5 min are also protected from infection with HSV-1. Though protection was lost over a period of 5 to 8 h, it was reinduced as rapidly as during the initial treatment. Below a 20 μM concentration of EB, cells gained protection in a serum-dependent manner, requiring bovine serum albumin (BSA) as a cofactor. Above 40 μM, EB coprecipitated with BSA under hypotonic conditions. Coprecipitates retained antiviral activity and released active peptide. NaCl (≥0.3 M) blocked coprecipitation without interfering with antiviral activity. As shown for β-galactosidase, EB below 20 μM acted as an enzyme inhibitor, whereas above 40 to 100 μM EB, β-galactosidase was precipitated as was BSA or other unrelated proteins. Pyrene fluorescence spectroscopy revealed that in the course of protein aggregation, EB acted like a cationic surfactant and self associated in a process resembling micelle formation. Both antiviral activity and protein aggregation did not depend on stereospecific EB interactions but depended strongly on the sequence of the peptide's hydrophobic tail. EB resembles natural antimicrobial peptides, such as melittin, but when acting in a nonspecific detergent-like manner, it primarily seems to target proteins.

Purpose: Vaccinia virus keratitis leading to blindness is a severe complication of smallpox vaccination. The clinical manifestations of vaccinia virus keratitis are similar to those of herpes simplex virus keratitis, a well-studied immunopathologic disease. Vaccinia virus keratitis is likely to involve an immunopathologic component, but little is known about the pathogenesis of the disease. The goal of this study was to determine type and kinetics of immune cell infiltration in the cornea during vaccinia virus keratitis., Methods: Rabbit eyes were trephined and inoculated with 1x10(5) pfu of the Dryvax strain of the vaccinia virus. On days 2, 4, 7, 10, 14, and 28 after infection, the animals were scored for clinical disease and eye sections were stained for B cells, CD4+ cells, CD8+ cells, and neutrophils. The eyelid, ciliary body, cornea, iris, iridocorneal angle, and choroid were examined., Results: Corneal vaccinia virus challenge resulted in the infiltration of B cells, CD4+ cells, CD8+ cells, and neutrophils into the cornea and eyelids. Neutrophils were the predominant cell type on days 2 and 3 after infection, whereas CD4+ cells were the predominant cell type detected in corneas on days 4 through 10. CD8+ cells and B cells peaked on day 10, but at lower levels than CD4+ cells and neutrophils., Conclusions: These results suggest that sequential migration of neutrophils, then CD4+ cells, plays an important role in vaccinia virus keratitis.

Purpose: The goal of this study was to use multiple quantitative disease measures to evaluate the effect of various viral inocula on the development of vaccinia keratitis in rabbits., Methods: Trephined eyes of female rabbits were infected with 10(4), 10(5), 10(6), or 10(7) plaque-forming units (pfu) of the Dryvax strain of the vaccinia virus and scored daily for disease for 14 days according to a modification of the MacDonald-Shadduck scoring system. Ocular viral titers and vaccinia-specific antibody titers were determined by plaque assay and ELISA, respectively., Results: The amount of virus used for infection affected the severity of disease, with 10(4) pfu eliciting milder keratitis after delayed onset compared with higher amounts of virus. At inocula above 10(5) pfu the course and severity of corneal disease was not significantly different. The time to reach peak titers was delayed in the 10(4) group but peak titers were similar in all groups. Severe conjunctival chemosis interfered with scoring in animals infected with 10(6) or 10(7) pfu. Virus-specific antibody titers were similar in all groups at day 14. Body weights decreased less than 10% in all groups., Conclusions: The course of vaccinia keratitis in rabbits paralleled that in humans. A viral inoculum of 10(5) pfu/eye was determined to be optimal for use in further studies of vaccinia keratitis.

Purpose. To determine the effects of COCH transgene expression on cultured human trabecular meshwork (HTM) cell morphology and on outflow facility (OF) in monkey organ cultured anterior segments (MOCAS). Methods. An adenoviral (Ad) vector expressing both cochlin (COCH) and green fluorescent protein (GFP) (AdCOCHGFP) or GFP alone (AdGFP) was used to transduce cultured HTM cells (multiplicity of transduction, 2.8 and 28). COCH transgene expression in transduced HTM cells and the culture medium was verified by Western blot analysis and immunofluorescence detection 5 days after transduction. MOCAS were used to test the effect of Ad vectors (2.8 x 10(10) viral particles per segment) on OF. The morphology of transduced MOCAS was evaluated by light microscopy. Results. Western blot analysis showed a viral vector dose-dependent expression of cochlin in transduced cells and the culture medium. There was no notable morphologic change in transduced cells. In MOCAS, cochlin expression was detectable in the medium by 3 days after transduction. A 35% decrease in OF in AdCOCHGFP-transduced MOCAS was detected after 3 days, decreasing by 76% after 12 days when compared to control segments injected with AdGFP. Anterior segment pressure (ASP) more than doubled (P < 0.05) in segments injected with AdCOCHGFP at 12 days after transduction. Light microscopy revealed normal angle structures in transduced segments. Conclusions. Ad vector delivery of the COCH transgene resulted in cochlin expression in HTM cells and MOCAS. Cochlin expression was effective in decreasing OF and increasing ASP in MOCAS, suggesting possible involvement of cochlin in IOP elevation in vivo. COCH gene delivery has potential for use in developing a glaucoma model.

In a chronic disease such as glaucoma, a therapy that provides a long lasting local effect with minimal systemic side effects, while circumventing the issue of patient compliance, is very attractive. The field of gene therapy is growing rapidly and ocular applications are expanding. Our understanding of the molecular pathogenesis of glaucoma is leading to greater specificity in ocular tissue targeting. Improvements in gene delivery techniques, refinement of vector construction methods, and development of better animal models combine to bring this potential therapy closer to reality.

Concerns about the possible use of Variola virus, the causative agent of smallpox, as a weapon for bioterrorism have led to renewed efforts to identify new antivirals against orthopoxviruses. We identified a peptide, EB, which inhibited infection by Vaccinia virus with an EC(50) of 15 microM. A control peptide, EBX, identical in composition to EB but differing in sequence, was inactive (EC50>200 microM), indicating sequence specificity. The inhibition was reversed upon removal of the peptide, and EB treatment had no effect on the physical integrity of virus particles as determined by electron microscopy. Viral adsorption was unaffected by the presence of EB, and the addition of EB post-entry had no effect on viral titers or on early gene expression. The addition of EB post-adsorption resulted in the inhibition of beta-galactosidase expression from an early viral promoter with an EC(50) of 45 microM. A significant reduction in virus entry was detected in the presence of the peptide when the number of viral cores released into the cytoplasm was quantified. Electron microscopy indicated that 88% of the virions remained on the surface of cells in the presence of EB, compared to 37% in the control (p<0.001). EB also blocked fusion-from-within, suggesting that virus infection is inhibited at the fusion step. Analysis of EB derivatives suggested that peptide length may be important for the activity of EB. The EB peptide is, to our knowledge, the first known small molecule inhibitor of Vaccinia virus entry.

The 773-residue ectodomain of the herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) has been resistant to the use of mutagenic strategies because the majority of the induced mutations result in defective proteins. As an alternative strategy for the identification of functionally important regions and novel inhibitors of infection, we prepared a library of overlapping peptides homologous to the ectodomain of gB and screened for the ability of the peptides to block infection. Seven of 138 15-mer peptides inhibited infection by more than 50% at a concentration of 100 microM. Three peptides (gB94, gB122, and gB131) with 50% effective concentrations (EC(50)s) below 20 microM were selected for further studies. The gB131 peptide (residues 681 to 695 in HSV-1 gB [gB-1]) was a specific entry inhibitor (EC(50), approximately 12 microM). The gB122 peptide (residues 636 to 650 in gB-1) blocked viral entry (EC(50), approximately 18 microM), protected cells from infection (EC(50), approximately 72 microM), and inactivated virions in solution (EC(50), approximately 138 microM). We were unable to discern the step or steps inhibited by the gB94 peptide, which is homologous to residues 496 to 510 in gB-1. Substitution of a tyrosine in the gB122 peptide (Y640 in full-length gB-1) reduced the antiviral activity eightfold, suggesting that this residue is critical for inhibition. This peptide-based strategy could lead to the identification of functionally important regions of gB or other membrane proteins and identify novel inhibitors of HSV-1 entry.

Purpose: The aim of this study was to elucidate the effects of the Rho-kinase inhibitor, H-1152, on cultured human trabecular meshwork (HTM) cells, TM morphology, and intraocular pressure (IOP) in rats., Methods: Cultured HTM cells were treated with H-1152. Changes in cell morphology and the organization of the actin cytoskeleton and focal adhesions were evaluated by microscopy and immunofluorescence. H-1152 was administered topically to the eyes of conscious rats, and IOP was measured with a commercially available tonometer before and after treatment. The eyes were enucleated 1 h after treatment, fixed, and processed for morphologic analysis by light and electron microscopy., Results: Exposure of the cultured HTM cells to 20 microM of H-1152 induced elongation and separation of cells, deterioration, and loss of actin stress fibers and focal adhesions within 2 h. Topical administration of H-1152 resulted in a significant decrease in IOP from 0.5 to 6 h, with the maximum IOP reduction of 28.1% at 1 h post-treatment (P < 0.001; n = 10). H-1152 caused an expansion of the intercellular spaces and loss of extracellular material in the juxtacanalicular region of the TM in rat eyes., Conclusions: The IOP-lowering effect of H-1152 in rat eyes is likely due to changes in TM-cell morphology, the actin cytoskeleton, and cellular adhesions in the conventional outflow pathway. H-1152 has potential as a new antiglaucoma medication.

Previously, it was shown that cationic alpha-peptides derived from the human immunodeficiency virus TAT protein transduction domain blocked herpes simplex virus type 1 (HSV-1) entry. We now show that cationic oligomers of beta-amino acids ("beta-peptides") inhibit HSV-1 infection. Among three cationic beta-peptides tested, the most effective inhibition was observed for the one with a strong propensity to adopt a helical conformation in which cationic and hydrophobic residues are segregated from one another ("globally amphiphilic helix"). The antiviral effect was not cell type specific. Inhibition of virus infection by the beta-peptides occurred at the postattachment penetration step, with a 50% effective concentration of 3 muM for the most-effective beta-peptide. The beta-peptides did not inactivate virions in solution, nor did they induce resistance to infection when cells were pretreated with the beta-peptides. The beta-peptides showed little if any toxicity toward Vero cells. These results raise the possibility that cationic beta-peptides may be useful antiviral agents for HSV-1 and demonstrate the potential of beta-peptides as novel antiviral drugs.

Gene delivery has potential for treating ocular disease and a number of delivery systems have been tested in animal models. However, several viral vectors have been shown to trigger undesirable transient inflammatory responses in the eye. Previously, it was shown that an attenuated Herpes simplex virus vector (hrR3) transduced numerous cell types in the anterior and posterior segments of monkey eyes, but this was accompanied by inflammation. In the retina, retinal pigment epithelial cells were the predominant cell type transduced by hrR3. IL-6 is an important pro-inflammatory cytokine and may play a role in the response to the hrR3 vector. Infection of human ARPE-19 cells with hrR3 resulted in increased IL-6 expression and secretion 3-4h post-infection. In the presence of acyclovir (70 microM) or in cells infected with UV-inactivated hrR3, IL-6 was not up-regulated indicating viral replication was required. Expression of the HSV-1 alpha and beta genes may be necessary but was not sufficient for NF-kappaB activation and IL-6 up-regulation. The translocation of NF-kappaB into the nucleus also occurred between 3 and 4h post-infection, coincident with increased IL-6 expression. Inhibition of NF-kappaB translocation by an Adenovirus vector expressing a dominant negative IkappaB (AdIkappaBam) inhibited IL-6 up-regulation, indicating that NF-kappaB plays a role in increasing IL-6 expression in APRE-19 cells. The hrR3 virus lacks viral ribonucleotide reductase (RR) activity, thus RR is not required for NF-kappaB activation or IL-6 up-regulation in ARPE-19 cells.

Purpose: To test the activity of a synthetic theta-defensin, retrocyclin (RC)-2, in a murine herpes simplex virus (HSV)-1 keratitis model., Methods: The in vitro antiviral activity of RC-2 against HSV-1 KOS was determined by yield reduction and viral inactivation assays. Efficacy in an experimental murine HSV-1 keratitis model was tested using pre- or postinfection treatment with 0.1% peptide in PBS with or without 2% methylcellulose. Viral titers in the tear film were determined by plaque assay., Results: RC-2 inhibited HSV-1 KOS in vitro with an EC(50) of 10 microM (~20 microg/mL) in yield-reduction assays, but was not directly virucidal. RC-106 (a less active analogue) did not inhibit HSV-1 KOS in culture. Incubating the virus with RC-2 or applying the peptide in 2% methylcellulose to the cornea before viral infection significantly reduced the severity of ocular disease, but postinfection treatment with 0.1% RC-2 in PBS with or without 2% methylcellulose did not. Viral titers were significantly reduced on some days after infection in the preincubation and prophylaxis groups., Conclusions: RC-2 was active against HSV-1 KOS in cultures and showed protective activity in vivo when used in a prophylactic mode, but the peptide showed limited activity in a postinfection herpes keratitis model. These findings support data obtained from experiments with HIV-1, HSV-2, and influenza A, indicating that RCs inhibit the entry of viruses rather than their replication.