Human herpesvirus 6: An emerging pathogen.

Infections with human herpesvirus 6 (HHV-6), a beta-herpesvirus of which two variant groups (A and B) are recognized, is very common, approaching 100% in seroprevalence. Primary infection with HHV-6B causes roseola infantum or exanthem subitum, a common childhood disease that resolves spontaneously. After primary infection, the virus replicates in the salivary glands and is shed in saliva, the recognized route of transmission for variant B strains; it remains latent in lymphocytes and monocytes and persists at low levels in cells and tissues. Not usually associated with disease in the immunocompetent, HHV-6 infection is a major cause of opportunistic viral infections in the immunosuppressed, typically AIDS patients and transplant recipients, in whom HHV-6 infection/reactivation may culminate in rejection of transplanted organs and death. Other opportunistic viruses, human cytomegalovirus and HHV-7, also infect or reactivate in persons at risk. Another disease whose pathogenesis may be correlated with HHV-6 is multiple sclerosis. Data in favor of and against the correlation are discussed

Immunotherapeutic efficacy of retargeted ohsvs designed for propagation in an ad hoc cell line

Our laboratory has pursued the generation of cancer‐specific oncolytic herpes simplex viruses (oHSVs) which ensure high efficacy while maintaining a high safety profile. Their blueprint included retargeting to a Tumor‐Associated Antigen, e.g., HER2, coupled to detargeting from natural receptors to avoid off‐target and off‐tumor infections and preservation of the full complement of unmodified viral genes. These oHSVs are “fully virulent in their target cancer cells”. The 3rd generation retargeted oHSVs carry two distinct retargeting moieties, which enable infection of a producer cell line and of the target cancer cells, respectively. They can be propagated in an ad hoc Vero cell derivative at about tenfold higher yields than 1st generation recombinants, and more effectively replicate in human cancer cell lines. The R‐335 and R‐337 prototypes were armed with murine IL‐12. Intratumorally‐administered R‐337 conferred almost complete protection from LLC‐ 1‐HER2 primary tumors, unleashed the tumor microenvironment immunosuppression, synergized with the checkpoint blockade and conferred long‐term vaccination against distant challenge tumors. In summary, the problem intrinsic to the propagation of retargeted oHSVs—which strictly require cells positive for targeted receptors—was solved in 3rd generation viruses. They are effective as immunotherapeutic agents against primary tumors and as antigen‐agnostic vaccines

Genotype of Immunologically Hot or Cold Tumors Determines the Antitumor Immune Response and Efficacy by Fully Virulent Retargeted oHSV

We report on the efficacy of the non-attenuated HER2-retargeted oHSV named R-337 against the immunologically hot CT26-HER2 tumor, and an insight into the basis of the immune protection. Preliminarily, we conducted an RNA immune profiling and immune cell content characterization of CT26-HER2 tumor in comparison to the immunologically cold LLC1-HER2 tumor. CT26-HER2 tumor was implanted into HER2-transgenic BALB/c mice. Hallmarks of R-337 effects were the protection from primary tumor, long-term adaptive vaccination directed to both HER2 and CT26-wt cell neoantigens. The latter effect differentiated R-337 from OncoVEXGM-CSF. As to the basis of the immune protection, R-337 orchestrated several changes to the tumor immune profile, which cumulatively reversed the immunosuppression typical of this tumor (graphical abstract). Thus, Ido1 (inhibitor of T cell anticancer immunity) levels and T regulatory cell infiltration were decreased; Cd40 and Cd27 co-immunostimulatory markers were increased; the IFNγ cascade was activated. Of note was the dampening of IFN-I response, which we attribute to the fact that R-337 is fully equipped with genes that contrast the host innate response. The IFN-I shut-down likely favored viral replication and the expression of the mIL-12 payload, which, in turn, boosted the antitumor response. The results call for a characterization of tumor immune markers to employ oncolytic herpesviruses more precisely

oHSV Genome Editing by Means of galK Recombineering

open8noThis work was supported by European Research Council (ERC) Advanced Grant number 340060, VII framework program to G. C.-F., by RFO (University of Bologna) to L.M. and T.G, and by Fondi Pallotti to T.G.Since the cloning of the herpes simplex virus (HSV) genome as BAC (bacterial artificial chromosome), the genetic engineering of the viral genome has become readily feasible. The advantage is that the modification of the animal virus genome is carried out in bacteria, with no replication or production of viral progeny, and is separated from the reconstitution or regeneration of the recombinant virus in mammalian cells. This allows an easy engineering of essential genes, as well. Many technologies have been developed for herpesvirus BAC engineering. In our hands the most powerful is galK recombineering that exploits a single marker (galK) for positive and negative selection and PCR amplicons for seamless modification in the desired genome locus. Here we describe the engineering of the HSV recombinant BAC 115 by the insertion of a heterologous cassette for the expression of murine interleukin 12 (mIL12) in the intergenic sequence between US1 and US2 ORFs.embargoed_20201017Laura Menotti, Valerio Leoni, Valentina Gatta, Biljana Petrovic, Andrea Vannini, Simona Pepe, Tatiana Gianni, Gabriella Campadelli-FiumeLaura Menotti, Valerio Leoni, Valentina Gatta, Biljana Petrovic, Andrea Vannini, Simona Pepe, Tatiana Gianni, Gabriella Campadelli-Fium

GFS, a preparation of Tasmanian Undaria pinnatifida is associated with healing and inhibition of reactivation of Herpes

BACKGROUND: We sought to assess whether GFS, a proprietary preparation of Tasmanian Undaria pinnatifida, has effects on healing or re-emergence of Herpetic infections, and additionally, to assess effects of GFS in vitro. Undaria is the most commonly eaten seaweed in Japan, and contains sulphated polyanions and other components with potential anti-viral activity. Herpes simplex virus type 1 (HSV-1) infections have lower reactivation rates and Herpes type 2 (HSV-2) infections have lower incidence in Japan than in the west. METHODS: Patients with active (15 subjects) or latent (6 subjects) Herpetic infections (HSV-1, 2, EBV, Zoster) were monitored for response to ingestion of GFS. GFS extract was tested in vitro for human T cell mitogenicity and anti-Herpes activity. RESULTS: Ingestion of GFS was associated with increased healing rates in patients with active infections. In addition, patients with latent infection remained asymptomatic whilst ingesting GFS. GFS extract inhibited Herpes viruses in vitro and was mitogenic to human T cells in vitro. CONCLUSIONS: Ingestion of GFS has inhibitory effects on reactivation and is associated with increased rate of healing after Herpetic outbreaks. GFS extract potently inhibited Herpes virus in vitro, and had mitogenic effects on human T cells

Dominance of variant A in Human Herpesvirus 6 viraemia after renal transplantation

<p>Abstract</p> <p>Background</p> <p>Human herpesvirus 6 (HHV-6), mostly variant B reactivation in renal transplant patients has been published by other authors, but the pathogenetic role of HHV-6 variant A has not been clarified. Our aims were to examine the prevalence of HHV-6, to determine the variants, and to investigate the interaction between HHV-6 viraemia, human cytomegalovirus (HCMV) infection and clinical symptoms.</p> <p>Methods</p> <p>Variant-specific HHV-6 nested PCR and quantitative real-time PCR were used to examine blood samples from renal transplant patients and healthy blood donors for the presence and load of HHV-6 DNA and to determine the variants. Active HHV-6 infection was proved by RT-PCR, and active HCMV infection was diagnosed by pp65 antigenaemia test.</p> <p>Results</p> <p>HHV-6 viraemia was significantly more frequent in renal transplant patients compared to healthy blood donors (9/200 vs. 0/200; p = 0.004), while prevalence of HHV-6 latency was not significantly different (13/200 vs. 19/200; p > 0.05). Dominance of variant A was revealed in viraemias (8/9), and the frequency of HHV-6A was significantly higher in active infections compared with latency in renal transplant patients (8/9 vs. 2/13; p = 0.0015). Latency was established predominantly by HHV-6B both in renal transplant patients and in healthy blood donors (11/13 and 18/19). There was no statistical significant difference in occurrence of HCMV and HHV-6 viraemia in renal transplant patients (7/200 vs. 9/200). Statistical analysis did not reveal interaction between HHV-6 viraemia and clinical symptoms in our study.</p> <p>Conclusions</p> <p>Contrary to previous publications HHV-6A viraemia was found to be predominant in renal transplant patients. Frequency of variant A was significantly higher in cases of active infection then in latency.</p

Herpes Simplex Virus Dances with Amyloid Precursor Protein while Exiting the Cell

Herpes simplex type 1 (HSV1) replicates in epithelial cells and secondarily enters local sensory neuronal processes, traveling retrograde to the neuronal nucleus to enter latency. Upon reawakening newly synthesized viral particles travel anterograde back to the epithelial cells of the lip, causing the recurrent cold sore. HSV1 co-purifies with amyloid precursor protein (APP), a cellular transmembrane glycoprotein and receptor for anterograde transport machinery that when proteolyzed produces A-beta, the major component of senile plaques. Here we focus on transport inside epithelial cells of newly synthesized virus during its transit to the cell surface. We hypothesize that HSV1 recruits cellular APP during transport. We explore this with quantitative immuno-fluorescence, immuno-gold electron-microscopy and live cell confocal imaging. After synchronous infection most nascent VP26-GFP-labeled viral particles in the cytoplasm co-localize with APP (72.8+/−6.7%) and travel together with APP inside living cells (81.1+/−28.9%). This interaction has functional consequences: HSV1 infection decreases the average velocity of APP particles (from 1.1+/−0.2 to 0.3+/−0.1 µm/s) and results in APP mal-distribution in infected cells, while interplay with APP-particles increases the frequency (from 10% to 81% motile) and velocity (from 0.3+/−0.1 to 0.4+/−0.1 µm/s) of VP26-GFP transport. In cells infected with HSV1 lacking the viral Fc receptor, gE, an envelope glycoprotein also involved in viral axonal transport, APP-capsid interactions are preserved while the distribution and dynamics of dual-label particles differ from wild-type by both immuno-fluorescence and live imaging. Knock-down of APP with siRNA eliminates APP staining, confirming specificity. Our results indicate that most intracellular HSV1 particles undergo frequent dynamic interplay with APP in a manner that facilitates viral transport and interferes with normal APP transport and distribution. Such dynamic interactions between APP and HSV1 suggest a mechanistic basis for the observed clinical relationship between HSV1 seropositivity and risk of Alzheimer's disease

Structural characteristics and antiviral activity of multiple peptides derived from MDV glycoproteins B and H

<p>Abstract</p> <p>Background</p> <p>Marek's disease virus (MDV), which is widely considered to be a natural model of virus-induced lymphoma, has the potential to cause tremendous losses in the poultry industry. To investigate the structural basis of MDV membrane fusion and to identify new viral targets for inhibition, we examined the domains of the MDV glycoproteins gH and gB.</p> <p>Results</p> <p>Four peptides derived from the MDV glycoprotein gH (gHH1, gHH2, gHH3, and gHH5) and one peptide derived from gB (gBH1) could efficiently inhibit plaque formation in primary chicken embryo fibroblast cells (CEFs) with 50% inhibitory concentrations (IC₅₀) of below 12 μM. These peptides were also significantly able to reduce lesion formation on chorioallantoic membranes (CAMs) of infected chicken embryos at a concentration of 0.5 mM in 60 μl of solution. The HR2 peptide from Newcastle disease virus (NDVHR2) exerted effects on MDV specifically at the stage of virus entry (i.e., in a cell pre-treatment assay and an embryo co-treatment assay), suggesting cross-inhibitory effects of NDV HR2 on MDV infection. None of the peptides exhibited cytotoxic effects at the concentrations tested. Structural characteristics of the five peptides were examined further.</p> <p>Conclusions</p> <p>The five MDV-derived peptides demonstrated potent antiviral activity, not only in plaque formation assays in vitro, but also in lesion formation assays in vivo. The present study examining the antiviral activity of these MDV peptides, which are useful as small-molecule antiviral inhibitors, provides information about the MDV entry mechanism.</p

Herpesviruses carrying a Brainbow cassette reveal replication and expression of limited numbers of incoming genomes

Whether all the infectious herpesvirus particles entering a cell are able to replicate and/or express their genomes is not known. Here, we developed a general method to determine the number of viral genomes expressed in an infected cell. We constructed and analysed fluorophore expression from a recombinant pseudorabies virus (PRV263) carrying a Brainbow cassette (Cre-conditional expression of different fluorophores). Using three isogenic strains derived from PRV263, each expressing a single fluorophore, we analysed the colour composition of cells infected with these three viruses at different multiplicities. We estimate that fewer than seven incoming genomes are expressed per cell. In addition, those templates that are expressed are the genomes selected for replication and packaging into virions. This finite limit on the number of viral genomes that can be expressed is an intrinsic property of the infected cell and may be influenced by viral and cellular factors