Herpes simplex virus type-1(HSV-1) oncolytic and highly fusogenic mutants carrying the NV1020 genomic deletion effectively inhibit primary and metastatic tumors in mice

Background. The NV1020 oncolytic herpes simplex virus type-1 has shown significant promise for the treatment of many different types of tumors in experimental animal models and human trials. Previously, we described the construction and use of the NV1020-like virus OncSyn to treat human breast tumors implanted in nude mice. The syncytial mutation gKsyn1 (Ala-to-Val at position 40) was introduced into the OncSyn viral genome cloned into a bacterial artificial chromosome using double-red mutagenesis in E. coli to produce the OncdSyn virus carrying syncytial mutations in both gB(syn3) and gK(syn1). Results. The OncdSyn virus caused extensive virus-induced cell fusion in cell culture. The oncolytic potential of the OncSyn and OncdSyn viruses was tested in the highly metastatic syngeneic mouse model system, which utilizes 4T1 murine mammary cancer cells implanted within the interscapular region of Balb/c mice. Mice were given three consecutive intratumor injections of OncSyn, OncdSyn, or phosphate buffered saline four days apart. Both OncSyn and OncdSyn virus injections resulted in significant reduction of tumor sizes (p \u3c 0.05) compared to control tumors. Virus treated mice but not controls showed a marked reduction of metastatic foci in lungs and internal organs. Mouse weights were not significantly impacted by any treatment during the course of the entire study (p = 0.296). Conclusion. These results show that the attenuated, but highly fusogenic OncSyn and OncdSyn viruses can effectively reduce primary and metastatic breast tumors in immuncompetent mice. The available bac-cloned OncSyn and OncdSyn viral genomes can be rapidly modified to express a number of different anti-tumor and immunomodulatory genes that can further enhance their anti-tumor potency. © 2008 Israyelyan et al; licensee BioMed Central Ltd

Glycoprotein B of human herpesvirus 8 is a component of the virion in a cleaved form composed of amino- and carboxyl-terminal fragments

Human herpesvirus 8 (HHV-8) or Kaposi\u27s sarcoma-associated herpesvirus (KSHV) is the only known human member of the Rhadinovirus genus of the gammaherpesvirus subfamily. Antibodies against peptides representing portions of the amino-and carboxyl-termini of HHV-8 gB were produced and used to detect gB expression in Vero cells transfected with the gB gene, in the HHV- 8-harboring cell line, BCBL-1, and in purified virions. Expression of gB was detected in approximately 3% of uninduced BCBL-1 cells, while up to 30% of the cells expressed gB after 12-O-tetradecanoylphorbol-13-acetate (TPA) induction of virus replication. Indirect immunofluorescence assays and confocal microscopy showed that gB was distributed throughout the cytoplasm of BCBL-1 cells and transfected Vero cells. Immunoblot analyses of virion preparations revealed the presence of full-length as well as two smaller than full-length gB-derived species corresponding to the amino- and carboxy- terminal portions of gB, respectively. Biochemical analysis of the gB carbohydrate moieties using glycosylation inhibitors revealed that gB contained N-linked oligosacharides of the high-mannose type, characteristic of precursor carbohydrate chains added in the endoplasmic reticulum. (C) 2000 Academic Press

Role of the Na\u3csup\u3e+\u3c/sup\u3e,K\u3csup\u3e+\u3c/sup\u3e Pump in Herpes Simplex Type 1-Induced Cell Fusion: Melittin Causes Specific Reversion of Syncytial Mutants with the Syn1 Mutation to Syn\u3csup\u3e+\u3c/sup\u3e (Wild-Type) Phenotype

To evaluate the importance of the Na+,K+ pump and ionic gradients in virus-induced cell fusion, we investigated the effects of melittin, a 26 amino acid bioactive peptide found in honey bee venom, on cell fusion caused by HSV-1 syncytial mutants. Melittin inhibited fusion of Vero cells caused by HSV-1 mutant viruses mP(MP), KOS (syn20) and KOS (FFV3) containing the syncytial mutation syn1 in glycoprotein K. However, it did not affect cell fusion caused by mutants HFEM(tsB5) or KOS amb 1511-7 with mutations in glycoprotein B. Melittin caused specific reversion of syn1 mutant virus plaques to syn+ (wild-type) plaque morphology, and inhibited virus adsorption and penetration. It also inhibited the Na+,K+ pump activity, and the binding of 3H-ouabain to the Na+,K+ pump of infected Vero cells. The Na+,K+ pump activity of infected Vero cells in comparison to mock-infected cells was significantly decreased. Ouabain, a specific inhibitor of the Na+,K+ pump, inhibited fusion of Vero cells caused by all syncytial virus strains. © 1993 Academic Press. All rights reserved

Specific antigens of Chlamydia pecorum and their homologues in C psittaci and C trachomatis

Objective - To analyze the antigenic cross reactivity of various proteins of strains of Chlamydia pecorum, C psittaci, and C trachomatis. Samples and Procedures - Strains FC-Stra and LW-613 of C pecorum; strains B577, Fitz-9, and 6BC of C psittaci, and strain LGV-2 of C trachomatis were studied. Strains of C pecorum were propagated in Georgia bovine kidney cells, and other chlamydial strains were propagated in L cells or Georgia bovine kidney cells. Partially purified chlamydial elementary bodies propagated in RAG cells, a BALB/c cell line cloned from a renal adenocarcinoma of BALB/c mice, were used to immunize BALB/c mice for production of monoclonal antibodies. Rabbits were inoculated with yolk sack-propagated, purified elementary bodies to produce polyclonal antisera. The reaction of monoclonal antibodies and polyclonal antisera with chlamydial proteins was analyzed by use of immunoblot techniques. Results - Two monoclonal antibodies reacted with a 90-kd protein of C psittaci and a 94-kd protein of C pecorum strains. One monoclonal antibody reacted strongly with a 67-kd protein of C pecorum and strain B577 of C psittaci, but weakly with proteins of strains 6BC and LGV-2. Another monoclonal antibody reacted with a 46-kd protein of 2 C pecorum strains and of strain B577 of C psittaci, but not with those of strains 6BC and LGV-2. Two monoclonal antibodies reacted with a 20-kd protein of C pecorum and a 22-kd protein of C psittaci and LGV-2 strains. Polyclonal antisera reacted similarly with the proteins identified by monoclonal antibodies in the various chlamydial strains. Conclusions - Reactions of several monoclonal antibodies with chlamydial antigens indicated that 67- and 46-kd proteins contain genus- and species-specific epitopes, respectively; a 94-kd protein of C pecorum is homologous to a 90-kd protein of C psittaci and C trachomatis strains; and a 20-kd protein of C pecorum corresponds to a 22-kd protein of C psittaci and C trachomatis

Protective immunity against lethal HSV-1 challenge in mice by nucleic acid-based immunisation with herpes simplex virus type-1 genes specifying glycoproteins gB and gD

DNA-based vaccines were employed to assess protective immunity against herpes simplex virus in experimental infections of hairless (strain SKH1) and BALB/c mice. Mice were vaccinated with plasmids containing the herpes simplex virus type-1 (HSV-1) glycoprotein B (gB) or D (gD) genes under the human cytomegalovirus immediate-early promoter control. Vaccines were injected intramuscularly (i.m.) or intraperitoneally (i.p.) as purified DNA alone or as formulations supplemented with different non-ionic block copolymers. Antibody responses were assessed by immunofluorescence and radioimmunoprecipitation assays. Mice inoculated with either gB or gD plasmid, alone or with non-ionic block copolymers CRL 1029 and CRL 1190, produced high levels of antibodies specific for gB or gD. Three weeks after the last vaccination, mice were challenged with a clinical HSV-1 isolate (ABGK-1) by inoculation of a shaved and subsequently scarified area between the third and fourth lumbar vertebrae. Mice immunised with either gD or gB plasmid alone or mixed with copolymers were protected against lethal HSV-1 challenge when immunisation was performed via the i.m. route. Immunisations given via the i.p. route induced humoral responses in some mice and protected the animals against lethal HSV-1 challenge only when the formulations contained copolymers. The BALB/c mouse model was shown to be as good a model as the hairless mouse model

Resolution of Genotypic and Phenotypic Properties of Herpes Simplex Virus Type 1 Temperature-Sensitive Mutant (KOS) tsZ47: Evidence for Allelic Complementation in the UL28 Gene

Herpes simplex virus type 1 (HSV-1) mutant tsZ47 was reported to be temperature sensitive for virus growth and transport of viral glycoproteins to the cell surface and to contain two different mutations (B. A. Pancake, D. P. Aschman, and P. A. Schaffer, (1983) J. Virol. 47, 568-585). However, we found that similar amounts of glycoproteins B, C and H were expressed at the cell surface at the permissive and non-permissive temperatures and in addition, ts Z47 virus contained only a single mutation. UL28-null virus, gCΔ7B, failed to complement tsZ47 in mixed infections and ts Z47 replicated in UL28 but not gB transformed cell lines. A ts lesion of tsZ47 was mapped within a 1333 bp region of the UL28 gene by marker-rescue using overlapping DNA fragments. DNA sequencing identified a C to T transversion resulting in an R to W amino acid change at UL28 amino acid position 531. Southern Blot analysis and transmission electron microscopy demonstrated that tsZ47, is defective in cleavage and encapsidation of viral DNA. Mutant virus ts1203 (C. Addison, F. J. Rixon, and V. G. Preston, (1990) J. Gen. Virol. 71, 2377-2384) that contains a mutation in the 5′ end of UL28, complemented tsZ47 in mixed infections. This suggests that allelic complementation may be occurring and UL28 may encode a protein with independently functioning domains, or that it participates in a multimer. © 1993 Academic Press. All rights reserved

Production of a rabbit anti-cockatiel immunoglobulin G and characterization of its cross-reactivities with immunoglobulin G of other psittacine species

The purpose of this work was to produce rabbit anti-cockatiel immunoglobulin G (IgG) and compare its cross-reactivity with sera from eight other psittacine birds: Quaker parakeet, budgerigar, green-wing macaw, blue-fronted Amazon parrot, eclectus parrot, African grey parrot, Patagonian conure, Moluccan cockatoo. Cockatiel IgG did not bind to protein A or G; therefore, these proteins could not be used in column chromatography to isolate the IgG. A combination of serum IgG precipitation by ammonium sulfate and yolk IgG extraction from egg was loaded in sodium dodecyl sulfate-polyacrylamide gel upon which the IgG was resolved by electrophoresis. The resolved IgG in sodium dodecyl sulfate-polyacrylamide gel was stained with Coomassie blue, cut, crushed in phosphate-buffered saline, and injected into rabbits. The rabbit anti-cockatiel IgG produced in this way reacted with a single protein in gel immunodiffusion assay with all nine psittacine bird sera but not with those of chicken and ostrich. Immunoelectrophoresis confirmed the cross-reactivity of different psittacine sera with the anti-cockatiel IgG serum but not with ostrich and chicken Sera. This antiserum detected antibody responses in sera from cockatiels vaccinated against chlamydial major outer membrane protein in an immunoblot assay

Thalidomide suppressed the growth of 4T1 cells into solid tumors in Balb/c mice in a combination therapy with the oncolytic fusogenic HSV-1 OncdSyn

Purpose: The anti-tumor properties of thalidomide or in combination with an oncolytic herpes virus (OncdSyn) was investigated in a mouse model of human breast cancer. Methods: To determine if thalidomide could act alone, 4T1 cells were injected into Balb/c mice. Tumors were sized, and the mice were fed chow or chow-containing thalidomide. After 4 days the tumor volumes were compared. To determine if thalidomide could act with the virus, tumors of mice were injected with phosphate buffered saline (PBS), or fed thalidomide with injections of PBS, or fed thalidomide with injections of OncdSyn, or received injections of OncdSyn. Results: Thalidomide alone suppressed tumor growth. The most significant treatment occurred in thalidomide-fed-OncdSyn-injected mice. Compared to PBS controls, there was a significant difference in the number of metastatic nodes in the lungs. Conclusions: Thalidomide alone delayed tumor growth, but the combination of thalidomide with OncdSyn appeared to produce the best results. © 2009 Springer-Verlag

Complete genome analysis and virulence characteristics of the Louisiana West Nile virus strain LSU-AR01

West Nile virus (WNV) is a member of the Flaviriridae family, which can cause significant morbidity and mortality in birds, horses, and humans. The WNV-LSU-AR01 strain was isolated from a dead blue jay in Louisiana in 2001. Phylogenetic analysis using 75 full WNV genomes revealed that the LSU-AR01 strain belongs to a distinct subclade among the North American strains. The LSU-AR01 strain differed from the NY-99 prototypic strain by 26 nucleotides causing six amino acid changes. An asparagine-to-lysine change was located immediately proximal to a known CD8+T cell epitope in NS4B, while a glutamine-to-lysine change was located within a predicted CD8+T cell epitope in NS5. The LSU-AR01 strain caused pronounced neuronal necrosis, perivascular cuffing and gliosis in comparison to the NY-99-infected mice. These results suggest that the previously identified Connecticut strains may contain highly neurovirulent strains such as the LSU-AR01 that have spread in North America. © 2009 Springer Science+Business Media, LLC