High prevalence of anelloviruses in vitreous fluid of children with seasonal hyperacute panuveitis.

Seasonal hyperacute panuveitis (SHAPU) is a potentially blinding ocular disease occurring in Nepal that principally affects young children. Random amplification of partially purified vitreous fluid (VF)-derived nucleic acid revealed the presence of human anelloviruses in VF of SHAPU patients. In a comparative study of patients with different ocular pathologies, SHAPU patients were at highest risk of harboring anelloviruses in their eyes. The majority of SHAPU patients had multiple anelloviruses in their VF. The ocular anellovirus load in SHAPU and non-SHAPU patients did not differ and no SHAPU-specific anellovirus variant was detected. Analysis of paired serum and VF samples from SHAPU and non-SHAPU patients showed that the anellovirus detected in VF samples most likely originated from the systemic viral pool during viremia, potentially through breakdown of the blood-ocular barrier. The detection of anelloviruses in VF samples of uveitis patients, profoundly so in SHAPU patients, is imperative and warrants elucidation of its clinical significance

Islands of linkage in an ocean of pervasive recombination reveals two-speed evolution of human cytomegalovirus genomes

Human cytomegalovirus (HCMV) infects most of the population worldwide, persisting throughout the host's life in a latent state with periodic episodes of reactivation. While typically asymptomatic, HCMV can cause fatal disease among congenitally infected infants and immunocompromised patients. These clinical issues are compounded by the emergence of antiviral resistance and the absence of an effective vaccine, the development of which is likely complicated by the numerous immune evasins encoded by HCMV to counter the host's adaptive immune responses, a feature that facilitates frequent super-infections. Understanding the evolutionary dynamics of HCMV is essential for the development of effective new drugs and vaccines. By comparing viral genomes from uncultivated or low-passaged clinical samples of diverse origins, we observe evidence of frequent homologous recombination events, both recent and ancient, and no structure of HCMV genetic diversity at the whole-genome scale. Analysis of individual gene-scale loci reveals a striking dichotomy: while most of the genome is highly conserved, recombines essentially freely and has evolved under purifying selection, 21 genes display extreme diversity, structured into distinct genotypes that do not recombine with each other. Most of these hyper-variable genes encode glycoproteins involved in cell entry or escape of host immunity. Evidence that half of them have diverged through episodes of intense positive selection suggests that rapid evolution of hyper-variable loci is likely driven by interactions with host immunity. It appears that this process is enabled by recombination unlinking hyper-variable loci from strongly constrained neighboring sites. It is conceivable that viral mechanisms facilitating super-infection have evolved to promote recombination between diverged genotypes, allowing the virus to continuously diversify at key loci to escape immune detection, while maintaining a genome optimally adapted to its asymptomatic infectious lifecycle

HSV Neutralization by the Microbicidal Candidate C5A

Genital herpes is a major risk factor in acquiring human immunodeficiency virus type-1 (HIV-1) infection and is caused by both Herpes Simplex virus type 1 (HSV-1) and HSV-2. The amphipathic peptide C5A, derived from the non-structural hepatitis C virus (HCV) protein 5A, was shown to prevent HIV-1 infection but neither influenza nor vesicular stomatitis virus infections. Here we investigated the antiviral function of C5A on HSV infections. C5A efficiently inhibited both HSV-1 and HSV-2 infection in epithelial cells in vitro as well as in an ex vivo epidermal infection model. C5A destabilized the integrity of the viral HSV membrane. Furthermore, drug resistant HSV strains were inhibited by this peptide. Notably, C5A-mediated neutralization of HSV-1 prevented HIV-1 transmission. An in vitro HIV-1 transmigration assay was developed using primary genital epithelial cells and HSV infection increased HIV-1 transmigration. Treatment with C5A abolished HIV-1 transmigration by preventing HSV infection and by preserving the integrity of the genital epithelium that was severely compromised by HSV infection. In conclusion, this study demonstrates that C5A represents a multipurpose microbicide candidate, which neutralizes both HIV-1 and HSV, and which may interfere with HIV-1 transmission through the genital epithelium

High prevalence of anelloviruses in vitreous fluid of children with seasonal hyperacute panuveitis.

Seasonal hyperacute panuveitis (SHAPU) is a potentially blinding ocular disease occurring in Nepal that principally affects young children. Random amplification of partially purified vitreous fluid (VF)-derived nucleic acid revealed the presence of human anelloviruses in VF of SHAPU patients. In a comparative study of patients with different ocular pathologies, SHAPU patients were at highest risk of harboring anelloviruses in their eyes. The majority of SHAPU patients had multiple anelloviruses in their VF. The ocular anellovirus load in SHAPU and non-SHAPU patients did not differ and no SHAPU-specific anellovirus variant was detected. Analysis of paired serum and VF samples from SHAPU and non-SHAPU patients showed that the anellovirus detected in VF samples most likely originated from the systemic viral pool during viremia, potentially through breakdown of the blood-ocular barrier. The detection of anelloviruses in VF samples of uveitis patients, profoundly so in SHAPU patients, is imperative and warrants elucidation of its clinical significance

Antiviral efficacy of C5A is preserved in genital fluids.

<p>TZM cells (100,000 cells/mL) were exposed to JR-CSF (1 ng of p24) for 4 h together with increasing concentrations of C5A or control non-amphipathic C5A peptide diluted in various conditions including DMEM (without serum), seminal plasma (8 donors), cervical fluids (4 donors) or vaginal mucus (4 donors). Cells were then washed and infection was measured 48 h after infection by β-galactosidase activity. Data are expressed as IC₅₀ in µM. Results are representative of 3 independent experiments.</p

HSV enhances HIV-1 transmigration through the genital epithelial barrier.

<p>A. Model for the HIV-1 transwell assay. B. PGEC were seeded onto polycarbonate membrane transwells and cultured until formation of tight junctions was achieved. HIV-1 (wild-type JR-CSF, NL4.3 or gp160-deficient (ΔE) NL4.3) was then added to the apical surface of PGEC and amounts of transcytosed viruses were collected at various time points (2, 4, 8 and 16 h) and quantified by p24 ELISA of the lower chamber corresponding to the basal surface. Data are expressed in percentage of the original viral inoculum (top panel). Infectivity of transcytosed viruses: medium from the basal chamber was collected at different intervals of time, filtered, standardized for p24 content (20 pg), and added to TZM indicator cells. Infection was measured 48 h post-infection by determining levels of beta-galactosidase activity in cell lysates (bottom panel). Results are representative of the results of four independent experiments using PGEC derived from each of the 4 donors. Error bars represent standard deviations. C. PGEC isolated from 2 donors were incubated with HSV-1 or HSV-2 (MOI of 0.005). Two days post-infection, infection of PGEC was quantified by FACS by anti-HSV gD IgG immunostaining. D. PGEC seeded onto the transwells were exposed to HSV-1 or HSV-2 (MOI of 0.005) in the presence or absence of ACV (50 µmol/L). Two days post-infection, the integrity of the PGEC barrier was analyzed by measuring the paracellular passage of the extracellular marker inulin (top panel) as well as the TER of each monolayer (bottom panel) as we described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018917#pone.0018917-Bobardt2" target="_blank">[19]</a>.</p

C5A prevents the HSV-mediated disruption of the genital epithelial cell barrier.

<p>A. PGEC monolayers were exposed to HSV-1, HSV-2 (MOI of 0.005) or the nonionic detergent N9 (80 µg/mL) for 2 days. HIV-1 (wild-type JR-CSF) (1 ng of p24) was then added to the apical surface of PGEC and amounts of crossed viruses were collected at various time points (2, 4, 8 and 16 h) and quantified by p24 ELISA of the lower chamber corresponding to the basal surface. Data are expressed in percentage of the viral inoculum originally added to the upper chamber. B. Same as A, except that just prior to HSV addition, ACV (50 µmol/L) was added to PGEC monolayers. C. Same as A, except that just prior to HSV addition, wild-type C5A or control non-amphipathic C5A variant (5 µM) was added to PGEC monolayers.</p

C5A blocks HSV infection.

<p>A. Vero cells were seeded in a 24-well plate and infected with increasing concentrations (MOI from 0.0001 to 1) of HSV-1 (Syn 17), HSV-2 (333) or HSV-1-GFP. C5A (5 µM) or control DMSO was added immediately after adding viruses to target cells. Two days post-infection, HSV-infected cultures (MOI 0.1) were analyzed by microscopy and representative pictures are depicted. B. Two days post-infection, infection of Vero cells was quantified by FACS either by mouse anti-HSV gD IgG2a immunostaining (HSV-1 Syn 17 and HSV-2 333) or by GFP content (HSV-1-GFP). For the gD cell surface staining, cells were washed and trypsinized 3 h post-infection to remove the original inoculum, and rabbit FITC-anti-mouse IgG were used to quantify bound mouse anti-HSV gD IgG. C. Human corneal and conjunctival epithelial cells were exposed for two days with increasing concentrations of HSV-1-GFP together with C5A (5 µM) or control DMSO. Infection was scored by GFP content. D. Primary human DC isolated from two donors were incubated with HSV-1 or HSV-2 (MOI of 0.1) together with C5A (5 µM) or control DMSO. HSV infection was quantified by cell surface expression of HSV gD two days post-infection as described above. Error bars represent standard errors of triplicates. Results are representative of two independent experiments.</p

C5A requirements for its anti-HSV effect.

<p>Vero cells were seeded in a 24-well plate and infected with HSV-1-GFP (MOI of 0.1). Increasing concentrations (1 to 8 µM) of wild-type or C5A variants were added to cells immediately after virus addition. After 48 h, HSV infection was quantified by FACS by GFP content. Percentage of infection in the absence of peptide was arbitrarily fixed at 100. Error bars represent standard errors of duplicates. Results are representative of two independent experiments.</p

C5A inhibits infectivity of drug-resistant HSV isolates.

<p>The susceptibility of ACV-, and GCV-resistant HSV isolates was determined by real-time PCR (qPCR) assay. Briefly, Vero cells were inoculated with HSV isolates for 1 h at 37°C. Viral inoculum was removed and cells were incubated, in triplicate, with increasing concentrations of ACV (0.05 to 50 µmol/L), GCV (0.05 to 50 µmol/L) or C5A (0.1 to 50 µM). At 24 h after inoculation, the supernatant was discarded and cells were lysed. Cell lysates were subjected to qPCR as we described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018917#pone.0018917-Duan1" target="_blank">[16]</a>. Viral load was determined on the basis of a standard curve generated on a stock of HSV-1 strain McIntyre. The IC₅₀ (µmol/L) was defined as the concentration of antiviral drug that reduced viral copies by 50%, compared with what was observed for infected control cells to which no drug was added.</p