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To establish infection, a wide variety of pathogens, including viruses, have evolved a number of strategies to avoid immune elimination. Viruses have acquired and optimized molecules that interact with the host chemokine network in order to disrupt immune surveillance and defense of vertebrates, helping to promote cell entry, facilitating dissemination of infected cells, and evasion the immune response. Viral immunomodulators include ligands, chemokine receptors and chemokine-binding proteins (vCKBPs) functioning as either cell surface receptor mimics, ligand mimics, or secreted chemokine-binding proteins. vCKBPs specifically modulate chemokine gradient formation and ligand-receptor recognition when they have a potential to even completely block chemokine-mediated responses to viral infection. Members of only two virus families (Herpesviridae and Poxviridae) encode vCKBPs capable of sequestering host chemokines through either the chemokine receptor, GAG-binding pocket, or both, which may result in the inhibition of chemotaxis in vivo. Here, we focused on vCKBPs encoded by α-, β-, and γ-herpesviruses, of which several have been experimentally used as anti-inflammatory or anti-immune reagents in animal models. Current results suggest that vCKBPs could be used to regulate the activity of both chemokines and chemokine receptors for the treatment of infections such as AIDS, diseases such as arthritis, neurotrauma, inflammatory CNS disorders, atherosclerosis, transplant rejection, and metastatic spread and angiogenesis. Better understanding of vCKBPs biology will help evaluate, which human diseases related to chemokine network dysregulation might be effectively treated with these novel promising immunomodulatory drugs to enable the manipulation of chemokine functions and leukocyte trafficking. Keywords: herpesviruses; chemokine-binding proteins; chemokines; immunomodulation viral infection, chemokines and viral immunomodulators.

Infection of human MRC-5 cells and mouse NIH-3T3 cells with a murine gamma-herpesvirus (MuHV-4 strain 68; MHV-68) photoinactivated by visible light in the presence of methylene blue (MB) resulted in nonproductive infection and the appearance of morphologically transformed cells. Two stably transformed cell lines were derived from both of these cell types and were confirmed to contain both viral DNA and antigen. Next, a quiescent MHV-68 infection in MRC-5 and NIH-3T3 cells was established after cultivation at 41°C in the presence of phosphonoacetic acid. Following the exposure of quiescently infected cells to visible light for 120 s (5 times daily for 6 days) in the presence of MB, both MRC-5 and NIH-3T3 cells were observed to acquire transformed phenotypes. The cytopathic effect was observed in cells after 4-5 passages, after which the cells degenerated. However, when human interferon (IFN)-α and mouse IFN-β were added to the media of quiescently infected MRC-5 and NIH-3T3 cells during the photoinactivating procedure, 2 stable transformed cell lines containing both viral DNA and the antigen were obtained and resembled those attained following nonproductive infection with photoinactivated virus.

The M3 protein (M3) encoded by murine gammaherpesvirus 68 (MHV-68) is a unique viral immunomodulator with a high-affinity for a broad spectrum of chemokines, key mediators responsible for the migration of immune cells to sites of inflammation. M3 is currently being studied as a very attractive and desirable tool for blocking the chemokine signaling involved in some inflammatory diseases and cancers. In this study, we elucidated the role of M3 residues E70 and T272 in binding to chemokines by examining the effects of the E70A and T272G mutations on the ability of recombinant M3, prepared in Escherichia coli cells, to bind the human chemokines CCL5 and CXCL8. We found that the E70A mutation enhanced binding of M3 to CCL5 two-fold but had little effect on its binding to CXCL8. In contrast, the T272G mutation was found to be important for the thermal stability of M3 and significantly decreased M3's binding to both CCL5 (by about 4×) and CXCL8 (by about 5×). We also constructed in silico models of the wild-type M3-CCL5 and M3-CCL8 complexes and found substantial differences in their physical and chemical properties. M3 models with single mutation E70A and T272G suggested the role of E70 and T272 in binding M3 protein to chemokines. In sum, we have confirmed that site-directed mutagenesis could be an effective tool for modulating the blockade of particular chemokines by M3, as desired in therapeutic treatments for severe inflammatory illnesses arising from chemokine network dysregulation.

Murine gammaherpesvirus 68 (MHV-68) is a natural pathogen that infects murid rodents which serve as hosts for Dermacentor reticulatus and Ixodes ricinus ticks. For the first time, MHV-68 was detected in immature I. ricinus ticks feeding on lizards trapped in Slovakia. Later on, MHV-68 infection was detected in D. reticulatus and Haemaphysalis concinna ticks collected on vegetation, which supported the idea that ticks can acquire the virus from feeding on infected hosts. Here, we report MHV-68 infection, which was detected by nested PCR, in D. reticulatus and I. ricinus adult ticks and I. ricinus nymphs collected in five geographically isolated localities, in west, southwest, south and central Slovakia. Viral incidence in ticks was 46.7% (121/259) without considering the season, site of collection and tick species and their life stage. MHV-68 infection was detected in all five localities investigated and in both tick species. Here, for the first time, we report MHV-68 infection in I. ricinus nymphs collected from the vegetation. The finding of virus in ticks from five separated localities suggested that ticks became infected with MHV-68 via feeding on infected rodents; thus, this virus might be a newfound natural pathogen in ticks.

The black fly Prosimulium italicum Rivosecchi, distributed in the Apennines and Sicily, was described as a subspecies of Prosimulium hirtipes (Fries), based on a few morphological details. It subsequently was considered conspecific with P. hirtipes and the name was synonymized. Analyses of polytene chromosome banding patterns and sequences of mitochondrial DNA (COI and COII) revealed deep genetic divergence between P. italicum from Italy and P. hirtipes from northern and central Europe and confirmed the species status of P. italicum . Populations of P. italicum either lack chromosomal inversion IS-9 or carry it as an X-chromosome polymorphism, whereas all analyzed populations of P. hirtipes (Slovakia, Sweden, England, and Scotland) are fixed for IS-9 . The average K2P genetic distance was 3.7% between P. italicum and P. hirtipes from northern Europe (Sweden) and 4.3 % between P. italicum and P. hirtipes from central Europe (Slovakia). Cytogenetic analysis showed the presence of two cytoforms of P. hirtipes (‘A’ in Sweden and Slovakia and ‘B’ in England and Scotland) and two cytoforms of P. italicum (‘A’ in Sicily and ‘B’ in Campania and Basilicata), all of which differ in their sex chromosomes and autosomal polymorphisms, suggesting that P. hirtipes and P. italicum might each be a complex of cryptic species.

Human dermal fibroblasts and mouse NIH/3T3 cells acquired the transformed phenotype (‘criss-cross' pattern of growth) after infection with ultraviolet-irradiated murine gammaherpesvirus (MuHV-4 strain 68; MHV-68). These cells with changed phenotype could be serially cultured for 5-6 passages (35-40 days), and then they entered into crisis and most of them died. In a small number of cultures, however, foci of newly transformed cells appeared from which two stable cell lines were derived. After 6-9 cell culture passages of the MHV-68 transformed cell lines, MHV-68 DNA and virus antigen could be detected by PCR and immunofluorescence assay along with the disappearance of actin bundles, indicating that both transformed cell lines might be oncogenic.

M3 protein of murine gammaherpesvirus 68 (MHV-68) was identified as a viral chemokine-binding protein 3 (vCKBP-3) capable to bind a broad spectrum of chemokines and their receptors. During both acute and latent infection MHV-68 M3 protein provides a selective advantage for the virus by inhibiting the antiviral and inflammatory response. A unique mutation Asp307Gly was identified in the M3 protein of murine gammaherpesvirus 72 (MHV-72), localized near chemokine-binding domain. Study on chemokine-binding properties of MHV-72 M3 protein purified from medium of infected cells implied reduced binding to some chemokines when compared to MHV-68 M3 protein. It was suggested that the mutation in the M3 protein might be involved in the attenuation of immune response to infection with MHV-72. Recently, Escherichia coli cells were used to prepare native recombinant M3 proteins of murine gammaherpesviruses 68 and 72 (Pančík et al., 2013). In this study, we assessed the chemokine-binding properties of three M3 proteins prepared in E. coli Rosetta-gami 2 (DE3) cells, the full length M3 protein of both MHV-68 and MHV-72 and MHV-68 M3 protein truncated in the signal sequence (the first 24 aa). They all displayed binding activity to human chemokines CCL5 (RANTES), CXCL8 (IL-8), and CCL3 (MIP-1α). The truncated MHV-68 M3 protein had more than twenty times reduced binding activity to CCL5, but only about five and three times reduced binding to CXCL8 and CCL3 when compared to its full length counterpart. Binding of the full length MHV-72 M3 protein to all chemokines was reduced when compared to MHV-68 M3 protein. Its binding to CCL5 and CCL3 was reduced over ten and seven times. However, its binding to CXCL8 was only slightly reduced (64.8 vs 91.8%). These data implied the significance of the signal sequence and also of a single mutation (at aa 307) for efficient M3 protein binding to some chemokines.

Gammaherpesviruses-encoded replication and transcription activator (Rta) (ORF50) plays an essential role in the initiation of viral lytic gene expression and reactivation from latency. The Rta expression is influenced by many viral and cellular factors, including epigenetic modifications, mainly DNA methylation and histone modifications. Murine gammaherpesvirus 68 (MHV-68), belonging to the species Murid herpesvirus (MuHV-4), is widely used as a model to study human gammaherpesvirus infections in vitro as well as in vivo. Recent studies of the MHV-68 Rta promoter revealed the effect of DNA demethylation and histone acetylation, induced by the inhibitor of histone deacetylase trichostatin A (TSA), on the MHV-68 reactivation from latency. Two other strains of MuHV-4, MHV-72 and MHV-4556, possess several unique properties, which distinguish them from strain MHV-68. Recently discovered reduced capacity of MHV-72 and MHV-4556 to reactivate from latency may be related to different methylation/demethylation patterns of the promoter regulatory region of the Rta. Here, we investigated the epigenetic regulation of the Rta promoter of three murine gammaherpesvirus strains, MHV-68, MHV-72 and MHV-4556, during latency and reactivation in vivo. However, we did not find any differences between Rta of MHV-68, MHV-72 and MHV-4556 and its epigenetic regulation during lytic infection, latency and de novo infection after ex vivo and in vivo reactivation induced by TSA. We confirmed that the treatment with TSA successfully induced demethylation of the Rta promoter regions of all three studied strains. Moreover, we have shown that the primary sequence of Rta and its promoter is identical for all three strains.

The new, small cavernicolous species Helmablatta louisrothi gen. et sp. n. (Nocticolidae) from the Tan-Phu cave (Vietnam) is one of the most morphologically interesting cockroaches. The extremely modified upstanding tergal gland composite from three tergites and may serve for gripping the female head during copulation. This presumption is supported by the presence of a central big hook on tergite 8. Furthermore, both wing pairs are uncommonly adapted to help releasing sex pheromones without raising the wings. Histone 3 DNA-based maximum likelihood analyses indicate a recent origin and close phylogenetic relationship between Nocticola spp. and Helmablatta sp.—consistent with the Quaternary age of the source lava tubes.

Herpesviruses are a large group of DNA viruses infecting mainly vertebrates. Murine gammaherpesvirus 68 (MHV68) is often used as a model in studies of the pathogenesis of clinically important human gammaherpesviruses such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. This rodent virus appears to be geographically widespread; however, its natural transmission cycle is unknown. Following detection of MHV68 in field-collected ticks, including isolation of the virus from tick salivary glands and ovaries, we investigated whether MHV68 is a tick-borne virus. Uninfected Ixodes ricinus ticks were shown to acquire the virus by feeding on experimentally infected laboratory mice. The virus survived tick molting, and the molted ticks transmitted the virus to uninfected laboratory mice on which they subsequently fed. MHV68 was isolated from the tick salivary glands, consistent with transmission via tick saliva. The virus survived in ticks without loss of infectivity for at least 120 days, and subsequently was transmitted vertically from one tick generation to the next, surviving more than 500 days. Furthermore, the F1 generation (derived from F0 infected females) transmitted MHV68 to uninfected mice on which they fed, with MHV68 M3 gene transcripts detected in blood, lung, and spleen tissue of mice on which F1 nymphs and F1 adults engorged. These experimental data fulfill the transmission criteria that define an arthropod-borne virus (arbovirus), the largest biological group of viruses. Currently, African swine fever virus (ASFV) is the only DNA virus recognized as an arbovirus. Like ASFV, MHV68 showed evidence of pathogenesis in ticks. Previous studies have reported MHV68 in free-living ticks and in mammals commonly infested with I. ricinus, and neutralizing antibodies to MHV68 have been detected in large mammals (e.g., deer) including humans. Further studies are needed to determine if these reports are the result of tick-borne transmission of MHV68 in nature, and whether humans are at risk of infection.

Three strains of herpes simplex virus, K17syn- and HSZPsyn+ of type 1 (HSV-1) and USsyn- of type 2 (HSV-2), were photoinactivated in the presence of methylene blue and used to infect 3 cell lines, normal human lung tissue cells (MRC-5), mouse epithelial cells (NIH3T3), and human lung carcinoma cells (A549). The virus titer and phenotype of cells were evaluated to compare the characteristics of normal and carcinoma cells infected with non-syncytial (non-syn) and syncytial (syn) strains of herpes simplex viruses. We found that the cells of both normal cell lines infected with photoinactivated K17syn- and USsyn- but not HSZPsyn+ acquired transformed phenotype accompanied by the presence of virus. Surprisingly, the infection with photoinactivated viruses K17syn- and USsyn- but not HSZPsyn+ resulted in the suppression of the transformed phenotype of A549 cells. Using nested PCR, herpesviral DNA was identified in newly transformed cells and cells that lost the transformed phenotype. The effect of putative herpesvirus-related growth factors (HRGF) produced by cells infected with photoinactivated viruses was quantified and compared. Since methylene blue is currently used in phototherapy of herpetic lesions, these results raise the question of whether such therapy is risky to human health.

Murine herpesvirus 68 (MHV-68) is a natural pathogen that infects murid rodents, which serves as hosts for Ixodes ricinus ticks. For the first time, MHV-68 was detected in immature I. ricinus ticks feeding on Lacerta viridis lizards trapped in Slovakia, which supports the idea that ticks can acquire the virus from feeding on infected hosts. The recent discovery of MHV-68 infection and MHV-68 M3 gene transcripts in Dermacentor reticulatus ticks collected in Slovakia also supports this suggestion. Here, for the first time, we report MHV-68 infection, which was detected by nested PCR, in I. ricinus adults collected from the vegetation, and the viral load in infected ticks was determined by quantitative PCR. The viral incidence in ticks was 38.1% (21/55), and the viral load varied from 1.5 × 103 to 2.85 × 104 genome copies per tick. These results suggest that the I. ricinus ticks became infected with MHV-68 from biting infected rodents; thus, I. ricinus ticks may play a role in the spread of this virus in nature.

Summary. – This article is a brief summary of efforts to generate mutant herpesviruses for investigating and assigning gene functions of herpesviruses in replication and pathogenesis. While a full review of all herpesviruses is beyond the scope of this review, we focused our attention on the prototype of the herpesvirus subfamily – herpes simplex virus and murine gammaherpesvirus that serves as an excellent animal model to study human gammaherpesvirus pathogenesis. Furthermore, our present knowledge of essential, non-essential, and common genes of herpesviruses as well as of accessory genes that are currently being studied with the help of the bacterial artificial chromosome (BAC) system will also be discussed. This system facilitates the analysis of herpesviral genes with potential for use in gene therapy or as anti-cancer therapeutics.

Here we would like to provide a brief overview of the modern history of Marek΄s disease (MD) research with a focus on the most recent developments in experimental work and we will try to sum up their impact on the understanding of the biological properties of Marek΄s disease type 1 (MDV-1), the only representative of the Mardivirus genus causing fatal lymphoproliferative disease in poultry. We will also compare MDV-1 with other serologically-related poultry herpesviruses, Marek΄s disease virus type 2 (MDV-2) and herpesvirus of turkeys (HVT). Although MD was first described at the beginning of the last century, proper characterization of its biological impact on poultry production and utilization of molecular biology methods for detailed characterization of causative agent MDV-1 were introduced only in recent decades. However, many characteristics of MD infection, pathogenesis and vaccine protection mechanisms remain unclarified, though novel methods bring a challenge for better understanding of these unanswered questions.

Murine gammaherpesvirus 68 (MHV-68) is a natural pathogen of murid rodents, which serve as hosts to Haemaphysalis concinna ticks. The occurrence of MHV-68 was investigated in a total of 47 H. concinna adult ticks collected on the vegetation in Gabcikovo, situated in south-western Slovakia (47o54´0´´N, 17o35´0´´E), from May 2013 to May 2014. DNA from ticks was purified and screened by nested PCR targeting ORF50 of MHV-68 and the copy number of virus genome in ticks was determined by a real-time PCR assay specific for ORF65. The MHV-68 incidence in questing ticks was 38.3% (18/47) and the virus genome copy number per tick varied from 2x102 to 9.6x103. In this study, MHV-68 was documented for the first time in H. concinna ticks. Results expand previous data describing the occurrence of MHV-68 in Ixodes ricinus and Dermacentor reticulatus ticks collected in Slovakia, supporting the hypothesis that MHV-68 might be a newfound pathogen in ticks.

Viviparity evolved in bacteria, plants, ˃141 vertebrate lineages (ichthyosaurs, lizards, fishes, mammals, and others), and in 11 of 44 insect orders. Live-birth cockroaches preserved with brood sac (3D recovered two times optically) included Diploptera vladimir, Diploptera savba, Diploptera gemini spp.n., D. sp.1-2, and Stegoblatta irmgardgroehni from Green River, Colorado; Quilchena, Republic; McAbee, Canada; and Baltic amber, Russia (49, 54, and 45 Ma). They evolved from rare and newly evolved Blaberidae; they radiated circumtropically, later expanded into SE Asia, and have now spread to Hawaii and the SE USA. Association of autapomorphic characters that allow for passive and active protections from parasitic insects (unique wing origami pleating identical with its egg case-attacking wasp) suggest a response to high parasitic loads. Synchronized with global reorganization of the biota, morphotype destabilization in roaches lasted approximately 11-22 Ma, including both the adaptation of novel characters and the reduction of others. Thus, while viviparity can be disadvantageous, in association with new Bauplans and/or behaviors, it can contribute to the evolution of taxa with viviparous representatives that are slightly selectively preferred.

All records of blackflies (Simuliidae) from the territory of Croatia are summarized, including previously unpublished data. The blackfly fauna of Croatia consists of 28 species. Simulium (Nevermannia) angustitarse (Lundström), Simulium (Nevermannia) cryophilum (Rubtsov) complex, Simulium (Nevermannia) lundstromi (Enderlein), Simulium (Nevermannia) vernum Macquart complex, Simulium (Simulium) argyreatum Meigen, Simulium (Simulium) bezzii (Corti) complex, Simulium paraequinum Puri and Simulium pseudequinum Séguy are reported for the first time from Croatia. Information related to the ecoregions, in which species were found and specific species traits are given. Genus Prosimulium Roubaud is represented by one species only. Genus Simulium Latreille is represented by 27 species in six subgenera, with subgenus Simulium Latreille s. str. being most species rich (13 species) and subgenera Boophthora Enderlein and Trichodagmia Enderlein represented each by only one species. Compared to the neighboring countries, the Croatian species assemblage is most similar to the fauna of Slovenia and least similar to that of Italy. The relatively low number of species, presence of several species complexes and unclear identity of other species show that further research of blackflies in Croatia is needed.

The European black fly Simulium (Simulium) colombaschense (Scopoli), once responsible for as many as 22,000 livestock deaths per year, is chromosomally mapped, permitting its evolutionary relationships and pest drivers to be inferred. The species is 12 fixed inversions removed from the standard sequence of the subgenus Simulium. Three of these fixed inversions, 38 autosomal polymorphisms, and a complex set of 12 X and 6 Y chromosomes in 29 zygotic combinations uniquely characterize S. colombaschense and reveal 5 cytoforms: 'A' in the Danube watershed, 'B' in Italy's Adige River, 'C' in the Aliakmonas River of Greece, 'D' in the Aoös drainage in Greece, and 'E' in the Belá River of Slovakia. 'C' and 'D' are reproductively isolated from one another, and 'B' is considered a cytotype of 'A,' the probable name bearer of colombaschense. The species status of 'E' cannot be determined without additional collections. Three derived polytene sequences, based on outgroup comparisons, place S. colombaschense in a clade of species composed of the S. jenningsi, S. malyschevi, and S. reptans species groups. Only cytoforms 'A' and 'B' are pests. Within the Simuliidae, pest status is reached through one of two principal pathways, both of which promote the production of large populations of blood-seeking flies: (1) colonization of the world's largest rivers (habitat specialization) or (2) colonization of multiple habitat types (habitat generalization). Evolutionary acquisition of the ability to colonize large rivers by an ancestor of the S. jenningsi-malyschevi-reptans clade set the scene for the pest status of S. colombaschense and other big-river members of the clade. In an ironic twist, the macrogenome of S. colombaschense reveals that the name associated with history's worst simuliid pest represents a complex of species, two or more of which are nonpests potentially vulnerable to loss of their limited habitat.

Rickettsia slovaca, a member of spotted fever rickettsiae, is an agent of a mild human disease known as Tibola or Debonel. Using quantitative real-time PCR we identified the highest point of multiplication of wild and standard type (strain B) of R. slovaca on the second vs. the fourth day postinfection. Comparing both types of R. slovaca by transmission electron microscopy substantiated different cytopathological and morphological changes in infected cells and other differences, for example a slight shift of stages during the life cycle that presented in a variety of forms and localization of the studied Rickettsia within the infected cells. Our study provides a valuable insight into the pathogenicity and virulence of R. slovaca.

In this study, we investigated the medium of three cell lines transformed with murine herpesvirus 68 (MHV-68) in vitro and in vivo, 68/HDF, 68/NIH3T3, and S11E, for the presence of compounds resembling growth factors of some herpesviruses which have displayed transforming and transformed phenotype suppressing activity in normal and tumor cells. When any of spent medium was added to cell culture we observed the onset of transformed phenotype in baby hamster kidney cells (BHK-21) cells and transformed phenotype suppressing activity in tumor human epithelial cells (HeLa). In media tested, we have identified the presence of putative growth factor related to MHV-68 (MHGF-68). Its bivalent properties have been blocked entirely by antisera against MHV-68 and two monoclonal antibodies against glycoprotein B (gB) of MHV-68 suggesting viral origin of MHGF-68. The results of initial efforts to separate MHGF-68 on FPLC Sephadex G15 column in the absence of salts revealed the loss of its transforming activity but transformed phenotype suppressing activity retained. On the other hand, the use of methanol-water mobile phase on RP-HPLC C18 column allowed separation of MHGF-68 to two compounds. Both separated fractions, had only the transforming activity to normal cells. Further experiments exploring the nature and the structure of hitherto unknown MHGF-68 are now in the progress to characterize its molecular and biological properties.

Objective: The kinetics of immediate early (IE) and early (E) herpes simplex virus 1 (HSV-1) mRNA transcription was followed in explanted trigeminal ganglia from rabbits with established latency. Methods: The expression of IE and E mRNAs was first assessed in infected Vero cells by RT-PCR and then in explanted trigeminal ganglia by nested RT-PCR. Results: In infected Vero cells, IE mRNAs [for infected cell protein (ICP) 0, ICP4 and ICP27] were first detected 1–2 h post-inoculation (p.i.), peaking at 3 h p.i. The transcription of E mRNAs [for thymidine kinase (TK), RR1 and UL9], which were first detected from 3 h p.i., peaked between 5 and 10 h p.i. In explanted ganglia, the ICP0, ICP4 and ICP27 mRNAs were first detected after 4 h in culture. This was followed by the appearance of TK mRNA at 8 h and then by the UL9 mRNA, detected from 12 h post-explantation. A further E mRNA (RR1), as well as the late gC mRNA, were first observed after 24 h in culture. Moreover, ICP4 mRNA could be found in non-cultured ganglia. Conclusions: During reactivation of latent HSV-1 in explanted ganglia, the onset of ICP0 and ICP27 transcription at 4 h in culture was followed by TK transcription (at 8 h). Thus, in the rabbit reactivation model, ICP0 gene transcription rather than ICP4 transcription represents the relevant indicator of latency reactivation.

Lambda interferons (IFN-λ) are known to induce potent antiviral response in a wide variety of target cells. They activate the same intracellular signalling pathways and have similar biological activities as IFN-α/β, including antiviral activity, but signal via distinct receptor complex, which is expressed in a cell- and species-specific manner. IFN-λ was reported to induce in vitro marked antiviral activity against various RNA viruses, but corresponding data on DNA viruses are sparse. Therefore we examined the IFN-λ1 induced antiviral activity against two strains of herpes simplex virus 1, a highly pathogenic ANGpath and moderately pathogenic KOS. The antiviral response was determined in vitro in Vero cells, known as deficient in production of type I IFNs and in Vero E6 cells, responding to viral infection with abundant IFN-λ production, although deficient in production of type I IFNs. The results showed that IFN-λ1 induced in Vero cells higher antiviral activity against ANGpath strain than against KOS strain. In Vero E6 cells endogenous IFN-λ induced higher antiviral activity against ANGpath strain than against KOS strain, but because of the virus induction of IFN-λ expression the antiviral activity was detected later. The observed differences between the IFN-λ1-induced antiviral activities against viral strains of various pathogenicity suggest that virus attributes may play role in the antiviral state of cells induced by IFN-λ.

Murid herpesvirus 4 (MuHV 4) strain 68 (MHV-68) is a natural pathogen of murid rodents, which serves as hosts to Dermacentor reticulatus ticks. These ticks are known to transmit multiple pathogens, which can cause diseases in humans and animals. Recently, the detection of MHV-68 antibodies in the blood of animals living in the same biotope as virus-infected mice has suggested the role of ticks in pathogen circulation in nature. Herein, to identify MHV-68 in D. reticulatus ticks, DNA samples from 432 adults were collected at two sites in southwestern Slovakia from 2011 to 2014. Samples were examined by polymerase chain reaction (PCR), targeting ORF50 of MHV-68. Ignoring season and locality, we have found 25.9 % of the male and 44.9 % of the female ticks to be positive. Within ticks collected in Vojka, 40 % (125/312) became positive, at a rate of approximately 6.8 times higher in spring than in autumn (66 vs 9.7 %). In addition, in the spring, 1.4 times more females were positive than males. Within ticks collected in Gabcikovo, 23.3 % (28/120) became positive, with positive females being twice as frequent. The infecting virus was identified by analyzing amplified products via sequencing and restriction fragment length polymorphism (RFLP) analyses. Using an explantation/co-cultivation procedure, we examined the salivary glands, intestines, and ovaries of five females for live MHV-68. In all organs of two ticks, we identified a virus capable of replication in mammalian cells. This is the first report of MHV-68 detection in D. reticulatus ticks and of a live virus in their organs. Findings encourage further study to determine whether this potential arbovirus, found in salivary glands, is transmissible. It further supports the hypothesis regarding the mediating role of ticks in MHV-68 circulation in nature.

The genetic background of HSZP virus, an HSV1 strain with extensive passage history, was analyzed by parallel comparative sequencing of four relevant genes (UL27/gB, UL41/vhs, UL44/gC and UL53/gK) of HSZP and additional three selected viruses [strains ANGpath, strains KOS(a) and KOS(b) and the prototype strain 17]. Mutation at position 858 (His for Arg) in gB of HSZP was found to be responsible for giant cell formation (syn 3gB mutation) similarly as the 855 mutation (Val for Ala) in the gB of ANGpath. Nosyn 1gK mutations were detected in the UL53 gene either of HSZP or of ANGpath viruses. The reduced virulence of HSZP for adult mice after peripheral inoculation, similarly as that of KOS virus, seems to be related (at least in part) to numerous mutations in the gB ectodomain. Of these, two mutations located in the antigenic domain IV were the same in gBHSZP as well as in gBKOS (at amino acids 59 and 79), at least two (amino acids 313 and 553) were specific for gBKOS, while one mutation (Ser for Ala at position 108) was specific for gBHSZP. The abolished shutoff function of the HSZP virus was related to at least four out of six specific mutations seen in thevhs polypeptide (vhs HSZP) encoded by the UL41 gene, of which three (amino acids 374, 386, 392) were clustered in the semiconservative box A ofvhs HSZP (the truncation of which abrogates the inhibition provided by this protein) and one mutation (at amino acid 18) was situated in the highly conservative locus I ofvhs HSZP. In addition, the twovhs KOS specific mutations (amino acids 19 and 317) not found invhs HSZP, enhanced the early host shutoff function of thevhs KOS protein. Finally, gCHSZP had two specific mutations (amino acids 137 and 147) located in the antigenic domain II of gC, which is responsible for binding of HSV1 virions to the glycoso-aminoglycan (GAG) receptor. When expressed in Sf21 cells using the recombinatt baculovirus system (Bac-to-Bac), gCHSZP and gCKOS showed no essential antigenic differences.

In former studies, we described that the HSZP strain of herpes simplex virus type 1 (HSV-1) was defective with respect to the early shutoff of host protein synthesis but was effective at interfering with the early shutoff function of the HSV-1 strain KOS, even when heat-inactivated or neutralized by antibody. However, the HSZP strain failed to interfere when inactivated with zinc ions or purified from cells treated with 2-deoxy-D-glucose. In this study, we provide evidence that the ability of the purified low-pH inactivated (citrate buffer, pH 3.0) and gel-filtered (Sephadex G-25) HSZP virions to adsorb host cells was not significantly affected. However, their ability to induce interference with the early shutoff function of the superinfecting HSV-1 strain KOS was restricted. In comparison with native virus, up to eight times more low-pH inactivated HSZP virions were needed to interfere with the shutoff by strain KOS. The interference was not due to exclusion of strain KOS by HSZP at the level of adsorption and/or penetration. The restriction was partially overcome by treatment of the cells with polyethylene glycol after adsorption of the low-pH inactivated HSZP virions. This observation indicates that the direct fusion of the virion envelope of low-pH inactivated HSZP with the plasma cell membrane was predominantly hampered.

UNLABELLED Murine gammaherpesvirus 68 (MHV-68) -infected mouse is an animal model of gammaherpesvirus infection in man and domestic animals. Murine gammaherpesvirus 4556 (MHV-4556), isolated from Apodemus flavicollis ticks has been considered a close relative of MHV-68 but different in some features of infection in vitro and in vivo. Previous comparison of MHV-4556 with MHV-68 has revealed their diversity in immune evasion protein MK3. In this study, HindIII and EcoRI restriction profiles of the MHV-4556 genome disclosed absence of the deletion that has been identified previously at the left end of genomes of murine gammaherpesvirus 76 (MHV-76) and murine gammaherpesvirus Sumava (MHV-Sumava). A 22, 565 bp portion of MHV-4556 genome sequence was sequenced, analyzed and compared with that of MHV-68. Nucleotide sequences of 21 genes of MHV-4556 and deduced amino acid sequences revealed their identity to those of MHV-68 except for differences in 15 nucleotides and 8 amino acids in 5 genes and their proteins, respectively. Due to these differences, immune evasion protein M4 and structural proteins encoded by ORF8 (gB), ORF11 (p43), ORF26 and ORF52, respectively, are predicted to have a reduced hydrophilicity and surface exposure compard with their MHV-68 counterparts. These differences obviously contribute to some different pathogenetical features of these viruses and could explain the weaker immunogenicity of MHV-4556 in comparison with MHV-68. KEYWORDS murine gammaherpesvirus 4556; restriction analysis; partial genome sequence.

Murine gammaherpesvirus 68 (MHV-68)-infected mouse is a well known model for studies of Epstein-Barr virus (EBV)-related lymphoproliferative diseases (LPD). Murine gammaherpesvirus 72 (MHV-72) has been considered a close relative of MHV-68 but its replication in murine mammary gland cells and kinetics of infection of mice were found to be different. Pathological studies of a long-term-infection of mice revealed a similar or higher malignancy development rate in MHV-72-infected mice as compared with that of MHV-68. Previous comparison of MHV-72 with MHV-68 revealed their diversity in M3, MK3, and M7 genes encoding the chemokine-binding protein, immune evasion protein and glycoprotein 150, respectively. In this study, a portion (22,899 bp) of MHV-72 genome sequence was determined, analyzed and compared with that of MHV-68. Nucleotide sequences of 13 structural and 6 non-structural genes of MHV-72 and deduced amino acid sequences revealed their identity to those of MHV-72 except for differences in 9 nucleotides and 8 amino acids, occurring in 5 genes and their proteins. Due to these differences, 4 structural proteins encoded by ORF20, ORF26, ORF48, and ORF52, respectively, and a non-structural protein encoded by ORF4, all of MHV-72, are predicted to have altered hydrophilicity and surface exposure in comparison with their MHV-68 counterparts. These differences obviously contribute to some different pathogenetical features of these viruses and could explain the reduced immunogenicity of MHV-72 in relation to MHV-68, allowing MHV-72 to escape the host immune surveillance.

The MHV-68 (designed as Murid herpesvirus 4 (MuHV 4) strain 68) isolated from two rodents, Myodes glareolus and Apodemus flavicollis, is considered as a natural pathogen of free-living murid rodents. Recently, the detection of MHV antibodies in the blood of animals living in the same biotope as MHV-infected mice has suggested that ticks may have a role in the transmission of this pathogen. Ixodes ricinus is one the most abundant tick species in Europe known to transmit multiple pathogens causing human and animal diseases. In this study, nymphs and larvae feeding on 116 individuals of a temperate lizard species-the green lizard Lacerta viridis captured in the Slovak Karst National Park, were examined for MHV-68. The specific sequence of virion glycoprotein 150 was amplified in DNA individually isolated from I. ricinus ticks using single-copy sensitive nested polymerase chain reaction. MHV-68 was detected in ten of 649 nymphs and in five of 150 larvae, respectively. We found that 9.6% of green lizards fed at least one MHV-68-infected immature tick. Occurrence of MHV-68 within all ticks tested was 1.8%. This study is first to show that immature I. ricinus ticks feeding on free-living lizards in a Central European region could be infected with gammaherpesvirus (MHV-68), naturally infecting free-living murid rodents. Our results provide evidence supporting the hypothesis that ticks may play a mediating role in circulation of MHV-68 in nature.

Murine gammaherpesvirus 68 (MHV-68) contains gene-encoding M3 protein expressed during the acute and persistent phase of infection. This protein features a chemokine-binding activities (Parry et al., 2000; van Berkel et al., 2000). In this study, we demonstrated that the Murine gammaherpesvirus 72 (MHV-72) also contained M3 gene with the codon-changing mutation at the position 920 nt converting amino acid (aa) 307 Asp (GAC) to Gly (GGC). The mutation in the M3 protein was localized near chemokine-binding domain and was able to change the secondary structure of M3 protein. We examined the binding activities of M3 proteins of MHV-72 and MHV-68 to five human chemokines (CCL3, CCL5, CCL11, CCL2, and CXCL8). Binding activity of MHV-72 M3 protein to CCL5 as well as to CXCL8 reached only 11.1% (day 3 p.i.) to 20% (day 4 p.i.) of the activity detected for MHV-68 M3 protein. On the other hand, MHV-72 M3 protein bound to human cytokines CCL11 and CCL2 reached about 90% of the binding detected for MHV-68 M3 protein. The binding activity of both M3 proteins to human CCL3 was similar. These data implied that mutation identified in MHV-72 M3 protein might be involved in attenuation of immune response to infection with MHV-72.

Balb/c mice were immunized with the recombinant fusion protein gD1/313 (FpgD1/313 representing the ectodomain of HSV-1 gD), with the non-pathogenic ANGpath gE-del virus, with the plasmid pcDNA3.1-gD expressing full-length gD1 and with the recombinant immediate early (IE) HSV-1 protein ICP27. Specific antibodies against these antigens (as detected by ELISA) reached high titers with the exception of the DNA vaccine. High-grade protection against challenge with the virulent strain SC16 was found following immunization with the pcDNA3.1-gD plasmid and with the gE-del virus. Medium grade, but satisfactory protection developed after immunization with the FpgD1/313 and minimum grade protection was seen upon immunization with the IE/ICP27 polypeptide. A considerable response of peripheral blood cells (PBL) and splenocytes in the lymphocyte transformation test (LTT) was found in mice immunized with FpgD1/313, with the pcDNA3.1-gD plasmid and with the live ANGpathgE-del virus. For lymphocyte stimulation in vitro, the FpgD1/313 antigen was less effective than the purified gD1/313 polypeptide (cleaved off from the fusion protein); both proteins elicited higher proliferation at the 5 microg per 0.1 mL dose than at the 1 microg per 0.1 mL dose. The secretion of Th type 1 (TNF, IFN-gamma and IL-2) and Th type 2 (IL-4 and IL-6) cytokines was tested in the medium fluid of purified PBL and splenocyte cultures; their absolute values were expressed in relative indexes. The PBL from FpgD1/313 immunized mice showed increased secretion of both T(H)1 (TNF) as well as T(H)2 (IL-4) cytokines (7-10-fold, respectively). Splenocytes from FpgD1/313 immunized mice showed a significant (23-fold) increase in IL-4 production.

Murid herpesvirus 4 (MuHV-4) currently serves as a model for study of human gamma-herpesvirus pathogenesis. It codes for MK3 protein that similarly as K5 protein of Kaposi’s sarcoma-associated herpesvirus are members of a family of structurally related viral immune evasion molecules possessing RING-CH finger domain with ubiquitin ligase activity. Murine herpesvirus 72 (MHV-72) isolated from the same species of free-living small rodent is considered as closely related to Murine herpesvirus 68 (MHV-68). Studies on MHV-72, identified dissimilarity from MHV-68 in the sequence of glycoprotein 150 [K. Macakova, J. Matis, I. Režuchova, O. Kudela, H. Raslova, M. Kudelova, Virus Genes 26, 89–95 (2003)]. Murine herpesvirus 4556 (MHV-4556) is relatively new, till now, uncharacterised strain isolated from different murid species Apodemus flavicollis. We have therefore sequenced the MK3 gene of MHW-72 as well as of MHV-4556 to find out the evidence of their difference from that of MHV-68. We show here the unique nucleotide mutation in MHV-72 MK3 gene changing the codon at C-end of MK3 protein that was earlier predicted to function in interaction with TAP1/2. Furthermore, one from two nucleotide mutations found for MHV-4556 MK3 gene changed the codon that is localized at N-terminus of MK3 protein. MHV-4556-specific mutation was found within MK3 RING-CH finger domain known to be necessary for the ubiquitination of MHC class I proteins. Moreover, the latter established the new restriction site specific for MHV-4556.

Murine herpes virus (MHV), a natural pathogen originally isolated from free-living rodents, constitutes the most amenable animal model for human gamma herpesviruses. Based on DNA sequence homology, this virus was classified as Murid Herpesvirus 4 to subfamily Gammaherpesvirinae. Pilot studies in our laboratory, using mice inoculated by the intranasal route, showed that MHV infects macrophages, B lymphocytes, lung alveolar as well as endothelial cells. From the lungs the virus spreads via the bloodstream to spleen and bone marrow and via the lymphatics to the mediastinal lymph nodes. Similarly to other gamma herpesviruses, MHV established life-long latency maintained in host B lymphocytes and macrophages. An IM-like syndrome (per analogy to EBV) may develop during acute MHV infection, in which the atypical T/CD8+ lymphocytes eliminate viral DNA carrying B cells expressing the M2 latency associated protein. During latency, the MHV LANA (a KSHV LANA homologue) maintains the latent viral genome, assuring its copying and partition to new carrier cells in the course of division of the maternal cell. The nonproductive latency is turned onto virus replication by means of Rta protein. The chronic lymphoproliferative syndrome of unclear pathogenesis, which occurs in a certain part of latent MHV carriers, is related to the expression of gamma herpesvirus common latency-associated genes such as v-cyclin and/or to that of a virus-specific (M11/bcl-2) gene. This review attempts to summarize our knowledge concerning the function of MHV genes (either gamma herpesvirus common or MHV specific) related to immune evasion, latency and lymphoproliferation when highlighting the unsolved problems and/or controversial opinions.

Recombinant plasmids encoding either the full-length glycoprotein D (FLgD) or truncated gDs were constructed. The recombinant plasmids were expressed in Escherichia coli and BHK-21 cells. The strongest expression was obtained with the recombinant plasmid encoding a truncated gD which corresponded to the gD ectodomain. The cells transformed with this plasmid showed good exponential growth ensuring satisfactory yields of the expressed polypeptide in the form of the fusion protein. The fusion protein was biotinylated and efficiently purified. The shortest truncated gD, which contained the main continuous antigenic locus VII binding neutralization antibody and additional continuous antibody binding epitopes, still reacted with specific antibody as proven by immunoblot analysis. In addition, a shuttle vector for expression of FLgD in mammalian cells was constructed. This vector-transfected BHK-21 cells expressed gD for 40 days during 9 consecutive passages. The expression of gD began on day 2 and culminated at day 9 post transfection (p.t.).

The ori-binding protein (OBP), an early protein which is encoded by the herpes simples virus 1 (HSV-1) UL9 gene and initiates the replication of viral DNA, was expressed in Escherichia coli, purified on an avidin resin and used for preparation of a mouse antiserum to OBP (OBP antiserum). Expression and localization of OBP in HSV-1-infected Vero cells was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and indirect immunofluorescence test. RT-PCR revealed the presence of abundant UL9 transcripts from 3 to 12 hrs post infection (p.i). Traces of UL9 mRNA were detected already at 1.5 hr p.i. The OBP antiserum detected clumps of irregularly shaped structures in the nuclei of infected Vero cells first at 4 hrs p.i. These nuclear structures peaked at 5-6 hrs p.i. and later on (at 8-12 hrs p.i.) they changed into fine granules filling the whole nucleus.

Murine gammaherpesvirus 72 (MHV-72) is a virus of wild rodents and serves as a convenient small animal model to understand the pathogenesis of Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV8) infection. In laboratory mice MHV-72 causes an acute infection of lung epithelial cells and establishes the latency in B lymphocytes. In this study, we investigated athymic nude and immunocompetent mice for distribution of virus in organs after infection with MHV-72. Ten days following subcutaneous dorsal injection of nude mice, virus replicated in lungs, lymphoid organs, salivary glands and also in mammary glands. The virus titre decreased by day 21 post-infection in former tissues, but increased in mammary glands. Presence of virus DNA sequences was detected in the lymphoid and non-lymphoid tissues until the death of the animals (about 1 month post-infection). Infection of immunocompetent mice with MHV-72 induced replication of virus up to 42 days post-infection in mammary glands reaching the highest level of infectious virus at day 8 post-infection. These data show that there is latent infection in mice never detected before. Moreover, virus DNA was detected using nested PCR (by amplification of a portion of gp150 gene sequence) in the mammary glands and the milk of mouse mothers infected with MHV-72 2 days before delivery. We demonstrated the presence of virus DNA also in the milk removed from the stomach of non-infected newborn mice, which were nourished by infected mothers (wet-nurses) for 1 or 2 days. The failure to detect the virus DNA in newborn mice lungs confirmed that they did not become infected from wet-nurses by the intranasal route. This suggests that MHV may be naturally transmitted to newborn mice via breast milk.

In 1976, within a project on isolation of herpesviruses from small rodents in former Czechoslovakia, the mouse herpesvirus strain 68 (MHV-68) was isolated (Blaskovic et al., 1980). This virus was accepted by The International Committee on Taxonomy of Viruses (ICTV) as a new, so far unassigned species (member) of the Gammaherpesvirinae subfamily of the Herpesviridae family (Murphy et al., 1995). Besides MHV-68, four more isolates (MHV-60, MHV-72, MHV-76, and MHV-78) similar to MHV-68 were obtained in that field experiment in Slovakia. Later, three more isolates (MHV-Sumava from Bohemia and MHV-4556 and MHV-5682 from Slovakia) were obtained in other field experiments. All these isolates are in some properties similar but in others different from each other. Nevertheless, as their comparative genome sequence analysis is not yet available, we propose at present to regard all the abovementioned isolates as different isolates of the same virus and strain, i.e. MHV-68. It is not excluded that a more detailed characterization of these isolates in the future will lead to proposals of designating some of these isolates as new strains of the virus of concern. This review summarizes the up to date knowledge of various biological and physico-chemical properties of MHV-68. At least three isolates, MHV-68, MHV-72 and MHV-Sumava seem to be involved in malignant neoplasm development in mice. It should be stressed that the pathogenesis of the induced lymphoproliferative disease in mice is similar to that caused by Epstein-Barr virus (EBV) in humans.

Sequences of UL44 genes of strains HSZP, KOS and 17 of herpes simplex virus 1 (HSV-1) were determined and the amino acid sequences of corresponding glycoproteins (gC) were deduced. In comparison with the 17 strain, the HSZP strain showed specific changes in 3 nucleotides and in 2 amino acids (aa 139 and 147, both from Arg to Trp) in the antigenic locus LII. The change at aa 147 was situated within the GAG-binding epitope. In a similar comparison, KOS strain had changes in 3 nucleotides and 3 amino acids (aa 3, 14, and 300). The UL44 genes of HSZP and KOS strains were expressed in insect Sf-21 cells by means of the baculovirus (Bac-to-Bac) expression system. As shown by immunoblot analysis, both the recombinant baculoviruses (B1-HSZP and B6-KOS) expressed a glycosylated gC, the M(r) of which (116 K) was lower than that of gC synthesized in Vero cells (129 K) infected with strains HSZP or KOS. In addition, smaller gC-specific proteins (of apparent M(r) of 50-58 K and 98 K) corresponding to a non-glycosylated precursor polypeptide and/or incomplete forms of the partially glycosylated gC were found. When Balb/c mice were immunized with Sf-21 cells expressing gC, the recombinant gC-HSZP represented a more efficient immunogen possibly due to its stronger expression in these cells. The corresponding gC-HSZP antiserum reacted in enzyme-linked immunosorbent assay (ELISA) equally well with HSZP and KOS virion antigens and neutralized HSZP strain at a low titer. Both gC-HSZP and gC-KOS antisera detected the homologous as well as the heterologous gC antigens in Vero cells regardless whether infected with strains HSZP, KOS or 17, revealing the presence of gC from 6 to 16 hrs post infection (p.i.) in the cytoplasm, on the nuclear membrane and at the cell surface.

To investigate the potential of murine gammaherpesvirus 72 thymidine kinase (MHV-72-TK) to act as a suicide gene, we used a mammalian expression vector on rat fibroblastoid cells deficient in the cellular TK gene. Substrate specificity was assessed in vitro in cells with stable expression of MHV-72-TK. The Herpes simplex virus 1-TK (HSV-1-TK) was used as a reference suicide gene. Unlike HSV-1-TK modified cells, which were sensitive to ganciclovir (GCV) (IC50=9.7 μM), cells modified by MHV-72-TK did not show sensitivity to this drug. The use of 3′-azido-3′-deoxythymidine (AZT) and (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) did not affect the growth of cells expressing either MHV-72-TK or HSV-1-TK in the range of concentration used for AZT (0–375 μM) and for BVDU (0–50 μM). In contrast, 5′-fluoro-2′-deoxyuridine (5-FUdR) was extremely cytotoxic and effectively killed MHV-72-TK expressing cells (IC50 value 2.1 μM). This value was 16 times lower than that required to kill cells expressing HSV1-TK. To test whether the bystander effect between two heterologous cell types could be mediated by the MHV-72-TK/5-FUdR system in vitro, cells expressing MHV-72-TK were co-cultured with the tumour fibroblastoid cell line NAD for 48 hours before the drug (10.8 μM) was added. The cell mixtures contained various ratios of cells expressing MHV-72-TK (0 to 50% of total cells). Only 1% of MHV-72-TK-expressing cells were needed to enhance mouse tumour cell killing and to decrease the survival rate to 25.6%. The bystander effect was more pronounced when 10% of cells expressing MHV-72-TK were used, decreasing survival to 17.4%. In parallel, the same concentration of 5-FUdR dose only marginally inhibited tumour cell growth in the absence of exogenous TK activity (84% survival). These results demonstrate the efficiency of MHV-72-TK as a suicide gene when 5-FUdR is used as a pro-drug. When sequenced, MHV-72-TK proved to be identical to MHV-68 strain TK.

The nonpathogenic HSZP strain of HSV-1 induces large polykaryocytes due to a syn3 mutation (His for Arg at residue 858) in the C-terminal endodomain of glycoprotein B (gB) (40). We determined the nucleotide (nt) sequence of the UL27 gene specifying the gB polypeptide of HSZP (gBHSZP) and found 3 mutations in its ectodomain at aminoacids (aa) 59, 79 and 108. The ANGpath virus, which also has a syn3 mutation in the C-terminal endodomain of gB (Val for Ala at residue 855) is pathogenic for adult mice (39), but can be made nonpathogenic by replacing the gBANGpath gene by the corresponding gBKOS sequence (21). The gBANGpath had three ectodomain mutations (at aa 62, 77 and 285), while gBKOS had at least four ectomain mutations (aa 59, 79, 313, and 553). Two mutations (aa 59 and 79) in the latter, located in the variable antigenic site IV/D1 were common for gBKOS and gBHSZP. These together with the gBANGpath mutations at aa 62 and 77 create a cluster of 4 mutations in diverse region of the N-terminal part of gB (between aa 59-79), in which the gBs of pathogenic ANGpath and 17 viruses differ from the gBs of nonpathogenic HSZP and KOS viruses. The lower pathogenicity of KOS as related to gBKOS, is furthermore associated with the change of Ser to Thr at aa 313 (locus III/D2). The possibility is discussed that mutations in both above mentioned antigenic loci could result in higher immunogenicity of the corresponding antigenic epitopes, which, in turn, would contribute to the decreased virulence of HSZP and KOS viruses.

Glycoprotein K (gK) encoded by the UL53 gene is the ninth out of eleven HSV glycoproteins (gps). The precursor gK (pgK) is a transmembrane protein with four hydrophobic domains, which consists of 338 amino acids. The UL53 gene has two initiation codons: the upper overlaps with the UL52 ORF, while the lower is located 55 codons downstream and specifies a truncated precursor of the gK polypeptide. The UL53 gene and the upstream located UL52 gene have a common polyadenylation signal downstream from the UL53 stop codon so that the UL53 mRNA is completely nested within the UL52 transcript. The syn1 mutations in several KOSsyn mutants and in the MPsyn virus, which had been fine mapped to DNA coordinates 0.735-0.740, were later on located to the UL53 gene, especially to its portion which specifies the first 120 amino acids (aa) from the N-terminus (most frequently residue 40) and to a less precisely defined locus between aa 301-310 (close to the C-terminus). Point mutations in the N-terminal ectodomain of gK, which are related to syn formation, impair the putative ability of this region to down-regulate membrane fusion. The two N-glycosylated mannose core oligosaccharides are attached to the Asn residues of the gK polypeptide at positions 48 and 58, respectively. In infected cells, gK is localized mainly in the nuclear and endoplasmic reticulum (ER) membranes. It is not clear, whether gK becomes incorporated into the envelope of mature HSV particles. Studies with the insertion/deletion gK mutants showed the importance of gK for capsid envelopment, for the transportation and egress or virions from infected cells. It seems that gK has an essential role in virion egress, even though this glycoprotein acts in accord with gH and with another membrane protein encoded by the UL20 gene.

Parallel sequencing of UL53 gene of four strains of herpes simplex virus type 1 (HSV-1), two of which (HSZP and ANGpath) were of the syn phenotype while another two (KOS and 17) were of the non-syn phenotype, showed in three strains amino acid mutations unrelated to the already described syn1 glycoprotein K (gK) mutations (Dolter et al., 1994). The only mutations which altered encoded amino acids were found in strains HSZP (Gln to Arg at position 198) and ANGpath (Val to Ile at position 137). Both mutations were localised outside of the two mutation clusters suspected for affecting syncytium formation. In addition, a CG/GC variation was found at positions 245-246 and 669-670. These compressions affected three codons altering amino acids (aa) 82 (Cys or Ser), 223 (Me or Ile) and 224 (Leu or Val), respectively.

The UL41 gene of the HSZP strain of herpes simplex virus type 1 (HSV-1) defective with respect to the early shutoff of host protein synthesis was sequenced and compared with the corresponding HSV-1 strain KOS and 17 gene sequences. In comparison with strain 17, nine mutations (base changes) were HSZP specific, five KOS specific and four were common for both strains. Nine mutations caused codon changes. Three of these mapped to the nonconserved regions and the others to the conserved regions of the functional map of UL41 gene. One KOS specific mutation mapped to the region responsible for the binding of the virion host shutoff (vhs) protein to the alpha-transinducing factor (VP16). The possible relationship between mutations and host shutoff function is discussed. The nucleotide sequence data of the UL41 gene of HSZP and KOS have been submitted to the Genbank nucleotide database and have been assigned the accession numbers Z72337 and Z72338.

Strain HSZP of the herpes simplex virus type 1 (HSV-1) forms large giant cells in vitro. This property was found associated with a mutation that alters the codon CGC (in the strain KOS or 17 sequence) to CAC (in the HSZP sequence), changing the amino acid 857 from arginine to histidine in the cytoplasmic domain of the glycoprotein B (gB) polypeptide chain. Giant cell formation by ANGpath was attributed to a mutation that alters the codon GCC (in KOS and strain 17 sequences) to GTC (in ANGpath sequence) changing the amino acid 854 in the same (syn 3 ) region of the gB molecule. In contrast to the ANGpath virus, which is pathogenic (1 LD 50 4 PFU) for adult DBA/2 mice after peripheral inoculation, strain HSZP was never found to be lethal for adult mice. Whereas ANGpath-infected mice which survived acute infection frequently (79%) developed latency in the regional sensory ganglion (as proved by virus reactivation during explantation), latent HSZP reactivated in ganglion culture at a considerably reduced rate (21%). Only 10-day-old DBA/2 mice were sensitive to HSZP infection. In these, HSZP spread from the site of peripheral administration mainly by hematogenous route. The neural spread of HSZP in suckling DBA/2 mice was manifested by the involvement of vegetative neurons in the wall of the small intestine and in the retroperitoneal vegetative ganglia. We conclude the HSZP, a polykaryocyte-forming strain with a mutation in the syn 3 region II, shows limited neuroinvasity for mice after peripheral administration.

HSZP strain of herpes simplex virus type 1 (HSV-1)-unlike strains KOS, SC16 and ANGpath-established latency in the homolateral trigeminal ganglion of mice at a limited rate (21%) when tested by reactivation of latent virus in culture. If a nested polymerase chain reaction (PCR) for virus DNA detection was used, the positivity rate was 63-100%. The detection rate of HSZP DNA in acutely infected gangla did not differ from that of SC16 DNA provided that the sensitivity of PCR was below 20 pg of HSZP DNA per 0.5 microgram of total ganglionic DNA. The nested PCR assessed at least 200 fg of HSZP DNA per 0.5 microgram of ganglionic DNA.

A novel set of primers for polymerase chain reaction (PCR) which amplified the portion of US6 sequence coding for the main type-common neutralizing epitope of glycoprotein D (gD) was used for detection of herpes simplex virus (HSV) DNA in 44 cerebrospinal fluid (CSF) samples from 29 patients with clinical symptoms of viral meningitis or meningoencephalitis. The primers in question amplified the DNA of 9 out of 10 low-passage HSV-1 isolates and of 5 out of 10 HSV-2 low-passage isolates as well as the DNA of all laboratory strains examined when tested in the supernatant fluid of infected cells cultures. The PCR was positive in 5 CSF samples (taken on days 2, 4, 8, 10 and 56 after the onset of symptoms, but not later than day 8 after starting acyclovir (ACV) therapy) obtained from 4 patients with intrathecal antibody response. The PCR was repeatedly negative in CSF of 15 patients who had antibodies to HSV in serum and CSF, but did not show intrathecal antibody production. It was also negative in 10 patients who had no HSV antibodies in CSF. Our results confirmed that positive PCR for HSV DNA in the CSF is an indication for starting and/or continuing ACV therapy even in the absence of classical symptoms of HSV encephalitis.

A total of 139 rabbits was infected to the right scarified cornea with HSV type 1 strains Kupka, ANG, ANGpath and their gE defective (ANGpathI2-4), gC defective (ANGpathgC18), gC/gE negative (ANGpathCI-8) and gC/ICP4 deletion (ANGpathY1) mutants. Strains ANG, ANGpath, ANGpathgC18 and ANGpathY1 were, in contrast to the two gE negative mutants, highly lethal, but 79% of rabbits infected with the non-encephalitogenic Kupka strain survived. Strain Kupka and strain ANGpath gE-negative mutants I2-4 and gCI-8 were tested for their latency competence. While Kupka established latency in the homolateral trigeminal ganglia from 80% of infected rabbits, I2-4 did so in one of 10 animals only, and the gC/gE mutant gCI-8 was not harboured in any of infected animals in an inducible form. Significant correlation was found between shedding into the culture fluid of reactivated virus from the explanted ganglion and brain stem fragments at one hand and the presence of the viral DNA in these organs on the other hand as judged by spot blot hybridization with the HSV-1 strain 17 Kpn I fragments h and i to DNA extracts prepared from these organs. Hybridizations were predominantly negative with the DNA from the corresponding non-cultured organs, except in a few cases of non-cultured ganglion and brain stem from rabbits previously infected with the gE deletion mutants which displayed positive hybridization, although no virus reactivation could be observed in corresponding explants.

Both trigeminal ganglia, brain stem, and cornea from rabbits with established latent herpes simplex virus type 1 (HSV-1) infection were examined by explantation and by spot blot hybridization using strain 17 Kpn I fragments i, d, and h and the DNA extracted from above mentioned tissues. Correlation between positive hybridization and reactivation of infectious virus in the cultured explants was documented by enhanced hybridization with the DNA extracts from explanted ganglion samples. In addition, we found positive hybridization in some noncultured ganglion and brain stem samples which did not yield infectious virus by explantation. Keeping in mind the pitfalls of false positive hybridization, the results may indicate during latency the presence in neural tissues of HSV DNA sequences which did not spontaneously reactivate in culture.

A simple procedure for preparation of herpes simplex virus type 1 (HSV-1) subunit vaccine is described. The method is based on treatment of virus-infected cells with a nonionic detergent, removal of cell nuclei by low speed centrifugation, separation of viral nucleocapsids by high speed centrifugation through a sucrose cushion and, finally, precipitation of viral and cellular glycoproteins and proteins with ammonium sulphate (AS). Unlike to acetone precipitation, affinity chromatography on lectins or hydroxylapatite chromatography, AS precipitation repeatedly yielded the best and most reliable results as judged by relative protein content, antigenicity and immunogenicity of the final vaccine product.