The region of the herpes simplex virus type 1 LAT gene that is colinear with the ICP34.5 gene is not involved in spontaneous reactivation.

The goal of this report was to determine if the region of the LAT gene that is colinear with ICP34.5 (kb 6.2 to 7.1 of LAT) is involved in spontaneous reactivation of herpes simplex virus type 1. We inserted one copy of the ICP34.5 gene into the unique long region of a herpes simplex virus type 1 (strain McKrae) mutant lacking both copies of ICP34.5 (one in each viral long repeat) and the corresponding 917-nucleotide colinear portion of LAT (kb 6.2 to 7.1). Rabbits were ocularly infected with this mutant, and spontaneous reactivation relative to that for the wild-type virus and the original mutant was measured. As we previously reported, the original ICP34.5-deleted virus (d34.5) was significantly impaired for spontaneous reactivation and virulence (G. C. Perng, R. L. Thompson, N. M. Sawtell, W. E. Taylor, S. M. Slanina, H. Ghiasi, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler, J. Virol. 69:3033-3041, 1995). In contrast, we report here that restoration of one copy of ICP34.5 at a distant location completely restored the wild-type level of in vivo spontaneous reactivation, despite retention of the deletion in LAT (spontaneous reactivation rate = 0.3 to 1.4% for the ICP34.5 deletion mutant, 7.7 to 19.6% for the wild type, and 9 to 16.1% for virus with one copy of ICP34.5). Thus, the 917-nucleotide region of LAT from kb 6.2 to 7.1 was not involved in the LAT function required for wild-type spontaneous reactivation. We also found that restoration of a single ICP34.5 gene in a novel location did not restore wild-type virulence (rabbit death rate = 0% [0 of 15] for the original ICP34.5 deletion mutant, 8% [2 of 24] for the single-copy IPC34.5 virus, and 52% [14 of 27] for wild-type virus; P < 0.001 for one versus two copies of ICP34.5). It is likely that either two gene doses of ICP34.5 or its location in the long repeat is essential for full functionality of ICP34.5's virulence function. Furthermore, the ability of the single-copy ICP34.5 virus to reactivate at wild-type levels despite being significantly less virulent than wild-type virus separates the spontaneous reactivation phenotype from the virulence phenotype.     

An avirulent ICP34.5 deletion mutant of herpes simplex virus type 1 is capable of in vivo spontaneous reactivation.

The herpes simplex virus type 1 (HSV-1) ICP34.5 gene is a neurovirulence gene in mice. In addition, some ICP34.5 mutants have been reported to have a reduced efficiency of induced reactivation as measured by in vitro explantation of latently infected mouse ganglia. However, since spontaneous reactivation is almost nonexistent in mice, nothing has been reported on the effect of ICP34.5 mutants on spontaneous reactivation in vivo. To examine this, we have deleted both copies of the ICP34.5 neurovirulence gene from a strain of HSV-1 (McKrae) that has a high spontaneous reactivation rate in rabbits and used this mutant to infect rabbit eyes. All rabbits infected with the ICP34.5 mutant virus (d34.5) survived, even at challenge doses greater than 4 x 10(7) PFU per eye. In contrast, a 200-fold-lower challenge dose of 2 x 10(5) PFU per eye was lethal for approximately 50% of rabbits infected with either the wild-type McKrae parental virus or a rescued ICP34.5 mutant in which both copies of the ICP34.5 gene were restored. In mice, the 50% lethal dose of the ICP34.5 mutant was over 10(6) PFU, compared with a value of less than 10 PFU for the rescued virus. The ICP34.5 mutant was restricted for replication in rabbit and mouse eyes and mouse trigeminal ganglia in vivo. The spontaneous reactivation rate in rabbits for the mutant was 1.4% as determined by culturing tear films for the presence of reactivated virus. This was more than 10-fold lower than the spontaneous reactivation rate determined for the rescued virus (19.6%) and was highly significant (P < 0.0001, Fisher exact test). Southern analysis confirmed that the reactivated virus retained both copies of the ICP34.5 deletion. Thus, this report demonstrates that (i) the ICP34.5 gene, known to be a neurovirulence gene in mice, is also important for virulence in rabbits and (ii) in vivo spontaneous reactivation of HSV-1 in the rabbit ocular model, although reduced, can occur in the absence of the ICP34.5 gene.     

Determination and Health-Risk Evaluation of Nitroxynil Residues in the Edible Tissue of Cattle

A specific Polarographic method with a sensitivity of ≥ 2 jig/kg (ppb) has been used to determine the plasma and tissue concentrations of nitroxynil (NTX), which is used against Parafilaria bovicola in cattle. After treatment with the therapeutic dose on NTX (2 × 20 mg/kg b.w., s,c), there was an initial rapid decrease in the plasma concentration followed by a slower elimination phase. The plasma levels of NTX were 8 mg/kg (ppm) and 3 mg/kg after 6 weeks and 2 months, respectively. The muscle and other edible tissue from treated cattle contained 0.1–0.3 mg/kg NTX after 2 months, and jxg/kg amounts were still detectable 3 months after the injection. Based on available pharmacological and toxicological data, a 3-months withdrawal time for NTX in cattle is proposed.     

Retention of tumorigenicity in radioresistant progeny of cells surviving exposure to high doses of gamma irradiation

The cell tumorigenic ability and the cell clonogenicity in semi-solid medium of highly radioresistant variant cell line, PIC-20 (the progeny of djungarian hamster fibroblast cell line DX-TK- surviving acute exposure to 20 Gy of gamma-irradiation), were examined. In the absence of additional radiation, no differences between tested features of non-irradiated PIC-20 cells and parental DX-TK- cells were observed. On the contrary, after gamma-irradiation with high doses the essential differences in the properties of the examined cell lines were revealed. After exposure to 10 Gy the surviving fraction of PIC-20 cells was 20 times higher than that of the parental cells. Both irradiated and non-irradiated PIC-20 cells produced colonies of similar size. It is revealed that even after irradiation with doses of 5, 10 or 15 Gy, the PIC-20 cells kept their tumorigenicity as high as non-irradiated ones. In all these cases the 90-100% of animals had the tumour, with the average latent period of tumour appearance after inoculation being the same both for irradiated and non-irradiated PIC-20 cells. After irradiation of parental DX-TK- cells with the highest dose of 15 Gy, the amount animals with tumour decreased by 70% and the average latent period of tumour appearance increased fivefold as compared with that for non-irradiated DX-TK- cells. The data obtained indicate that PIC-20 is highly radioresistant cells, which are able to proliferate both in semi-solid medium and in an animal organism even after radiation exposure to high doses.     

Local Periocular Vaccination Protects against Eye Disease More Effectively Than Systemic Vaccination following Primary Ocular Herpes Simplex Virus Infection in Rabbits

Vaccination of experimental animals can provide efficient protection against ocular herpes simplex virus type 1 (HSV-1) challenge. Although it is suspected that local immune responses are important in protection against ocular HSV-1 infection, no definitive studies have been done to determine if local ocular vaccination would produce more efficacious protection against HSV-1 ocular challenge than systemic vaccination. To address this question, we vaccinated groups of rabbits either systemically or periocularly with recombinant HSV-2 glycoproteins B (gB2) and D (gD2) in MF59 emulsion or with live KOS (a nonneurovirulent strain of HSV-1). Three weeks after the final vaccination, all eyes were challenged with McKrae (a virulent, eye disease-producing strain of HSV-1). Systemic vaccination with either HSV-1 KOS or gB2/gD2 in MF59 did not provide significant protection against any of the four eye disease parameters measured (conjunctivitis, iritis, epithelial keratitis, and corneal clouding). In contrast, periocular vaccination with gB2/gD2 in MF59 provided significant protection against conjunctivitis and iritis, while ocular vaccination with live HSV-1 KOS provided significant protection against all four parameters. Thus, local ocular vaccination provided better protection than systemic vaccination against eye disease following ocular HSV-1 infection. Since local vaccination should produce a stronger local immune response than systemic vaccination, these results suggest that the local ocular immune response is very important in protecting against eye disease due to primary HSV-1 infection. Thus, for clinical protection against primary HSV-1-induced corneal disease, a local ocular vaccine may prove more effective than systemic vaccination.     

Cell invasion by Neisseria meningitidis requires a functional interplay between the focal adhesion kinase, Src and cortactin

Entry of Neisseria meningitidis (the meningococcus) into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK), which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK) blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells.     

Role of the herpes simplex virus helicase-primase complex during adeno-associated virus DNA replication

A subset of DNA replication proteins of herpes simplex virus (HSV) comprising the single-strand DNA-binding protein, ICP8 (UL29), and the helicase-primase complex (UL5, UL8, and UL52 proteins) has previously been shown to be sufficient for the replication of adeno-associated virus (AAV). We recently demonstrated complex formation between ICP8, AAV Rep78, and the single-stranded DNA AAV genome, both in vitro and in the nuclear HSV replication domains of coinfected cells. In this study the functional role(s) of HSV helicase and primase during AAV DNA replication were analyzed. To differentiate between their necessity as structural components of the HSV replication complex or as active enzymes, point mutations within the helicase and primase catalytic domains were analyzed. In two complementary approaches the remaining HSV helper functions were either provided by infection with HSV mutants or by plasmid transfection. We show here that upon cotransfection of the minimal four HSV proteins (i.e., the four proteins constituting the minimal requirements for basal AAV replication), UL52 primase catalytic activity was not required for AAV DNA replication. In contrast, UL5 helicase activity was necessary for fully efficient replication. Confocal microscopy confirmed that all mutants retained the ability to support formation of ICP8-positive nuclear replication foci, to which AAV Rep78 colocalized in a manner strictly dependent on the presence of AAV single-stranded DNA (ssDNA). The data indicate that recruitment of AAV Rep78 and ssDNA to nuclear replication sites by the four HSV helper proteins is maintained in the absence of catalytic primase or helicase activities and suggest an involvement of the HSV UL5 helicase activity during AAV DNA replication.     

Real-time impedance analysis of host cell response to meningococcal infection

Measuring cell proliferation and cell death during bacterial infection involves performing end-point assays that represent the response at a single time point. A new technology from Roche Applied Science and ACEA Biosciences allows continuous monitoring of cells in real-time using specialized cell culture microplates containing micro-electrodes. The xCELLigence system enables continuous measurement and quantification of cell adhesion, proliferation, spreading, cell death and detachment, thus creating a picture of cell function during bacterial infection. Furthermore, lag and log phases can be determined to estimate optimal times to infect cells.In this study we used this system to provide valuable insights into cell function in response to several virulence factors of the meningitis causing pathogen Neisseria meningitidis, including the lipopolysaccharide (LPS), the polysaccharide capsule and the outer membrane protein Opc. We observed that prolonged time of infection with pathogenic Neisseria strains led to morphological changes including cell rounding and loss of cell-cell contact, thus resulting in changed electrical impedance as monitored in real-time. Furthermore, cell function in response to 14 strains of apathogenic Neisseria spp. (N. lactamica and N. mucosa) was analyzed. In contrast, infection with apathogenic N. lactamica isolates did not change electrical impedance monitored for 48 h. Together our data show that this system can be used as a rapid monitoring tool for cellular function in response to bacterial infection and combines high data acquisition rates with ease of handling.