Frank J. Jenkins¹, Alyson M. Donoghue² and John R. Martin³

¹ Department of Pathology and Infectious Diseases and Microbiology, University of Pittsburgh, and Division of Behavioral Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213
² Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD. 20814
³ Food and Drug Administration, Division of Antiviral Drug Products, Rockville, MD 20857.

Received 07/30/96; Accepted 08/27/96; On-line 10/04/96

RESULTS

The construction, isolation and characterization of the viral recombinants RBMu2, RBMu3 and REMu1 have been described previously (4,5). Figure 1 illustrates the genomic structure of the recombinants and the TK- parent HSV-1(F) 305. The deletion of the inverted repeat sequences, and specifically, the a repeat sequences (12,13) has resulted in the viral genomes becoming 'frozen' in a single isomeric form. These genomes are unable to invert and thus viral DNA isolated from either virions or infected cells contains only the single isomeric arrangement.

Each recombinant was independently isolated and differs from the others in both the extent of the internal repeat sequences that have been deleted and in the isomeric form in which the viral genomes are frozen. Thus RBMu2 is frozen in the prototype form while RBMu3 and REMu1 are frozen in the inverted S and inverted LS forms respectively. All three recombinant viruses are missing approximately 14 Kbp of the internal repeats with each deletion differing by only a few hundred base pairs (4,5).

in vitro growth curves

Our earlier studies (4,5) indicated that the recombinant viruses RBMu2, RBMu3, and REMu1 produced viral titers in Vero and rabbit skin cells comparable to those obtained with HSV-1(F). These results were based on the analysis of viral titers from infected cell cultures. To more carefully examine whether the deletion of the internal repeat sequences has any effect on viral replication in cell culture, growth curves of HSV-1(F) and the recombinant viruses were determined in Vero cells. Analysis of the growth curves demonstrated that the recombinant viruses grow somewhat more slowly than HSV-1(F) (Fig. 3). In addition, the recombinant viruses produced viral titers within 1.5 logs of HSV-1(F) in both Vero and primary mouse embryo fibroblast cells (Jenkins and Martin, unpublished observations). There were no detectable differences in the appearance or size of the plaques produced by these viruses as compared to the parental viruses in any of the cell culture lines tested (data not shown).

Figure 3: Growth curves of HSV-1(F) and the recombinant viruses in Vero cells.

All three recombinant viruses were selected and isolated by their ability to grow on human 143 TK- cells in the presence of HAT medium (100 µM hypoxanthine, 400 µM aminopterin, 16 µM thymidine), and should be expressing a functional TK gene product (contained within the mini-Mu insertion). Previous reports from other laboratories have demonstrated that a functional TK gene plays an important role in HSV induced pathogenicity in the mouse (14-16). Cells infected with the viral recombinants expressed greater than 93% of the TK activity found in cells infected with HSV-1(F) at 6 hours post-infection (hpi)(data not shown).

Studies on neurovirulence

To determine the effect of the deletions in the recombinant virus genomes on neurovirulence in the mouse, it was first necessary to determine the PFU/LD50 ratios for the wild-type parent HSV-1(F). We initially used three separate routes of inoculation (i.p., i.c., and scarified corneas) to measure neurovirulence since it has been reported that different strains of HSV vary greatly in their neurovirulence depending upon the route of inoculation (17-20). As shown in Table 1, while HSV-1(F) is quite avirulent by i.p. and corneal inoculation with PFU/LD50 ratios greater than 2.3x10⁶ and 1.7x10⁷ plaque forming units (pfu) respectively, i.c. inoculation demonstrated a PFU/LD50 ratio of 31, a value which agrees with previously published reports (21). Based on these results, neurovirulence studies with the recombinant viruses were confined to i.c. inoculations.The PFU/LD50 ratios for the recombinant viruses inoculated i.c. are shown in Table 2. The PFU/LD50 ratio for RBMu2 was 1.38x10⁶, while RBMu3 and REMu1 exhibited ratios of 1.06x10⁵ and >2x10⁶ respectively. Thus, deletion of the internal repeat sequences has resulted in a dramatic increase, ranging from three to four logs, in the PFU/LD50 ratios of these three recombinants compared to that of the parental wild-type HSV-1(F) strain.

Studies on neuroinvasiveness

We define neuroinvasiveness as the ability of the virus to spread from a local site of infection (e.g. cornea) to the peripheral nervous system (trigeminal ganglion). To determine if the recombinant viruses were neuroinvasive, scarified corneas of 10 six week old BALB/c male mice were inoculated with 1x10⁶ pfu of either HSV-1(F), RBMu2, or RBMu3. Each day for 5 days following inoculation, two mice from each group were anesthetized and the eyes and trigeminal ganglia harvested. The tissue was processed for acute virus as described in Methods. Plaque assays were performed on each tissue sample with the results presented in Figure 4. The parental wild-type strain HSV-1(F) demonstrated a biphasic growth pattern in the eyes of the mice which agrees with previous reports using different strains of HSV-1 (22,23). Analysis of the trigeminal ganglion from these mice demonstrated the presence of infectious HSV-1(F) as early as day 2, reaching its peak titer by day 4. In contrast, the recombinants RBMu2 and RBMu3 demonstrated a greatly reduced amount of virus in the eyes, with a slight increase in viral titer past day 1. Since virus was detected through day 4, and a slight increase in viral titer was observed between days 1 and 4, we believe that the recombinants exhibit a limited replication in the corneas of infected mice. However, analysis of the trigeminal ganglia from mice inoculated with the recombinants failed to detect any infectious virus (data not shown), indicating that the recombinants were not transported, at least in an infectious form, to the trigeminal ganglion. Therefore, the deletion of the internal repeat sequences results in the virus becoming non-neuroinvasive. Further, while corneas inoculated with HSV-1(F) would demonstrate a severe keratitis by day 5 - 6 which would gradually heal by day 14, corneal inoculations with the recombinant viruses failed to show any visible signs of keratitis at any time after inoculation (data not shown).

Figure 4: Growth of HSV-1(F) and the recombinant viruses RBMu2 and RBMu3 in the eye and HSV-1(F) in the trigeminal ganglion of mice.

Studies on latency

Two methods for studying HSV-induced latency involve either corneal or i.c. inoculations followed by the reactivation of latent virus from the trigeminal ganglia. Since the recombinant viruses were found to be non-neuroinvasive by corneal inoculation, it is unlikely that the viruses would establish a latent infection in trigeminal ganglia following corneal inoculation. This hypothesis is supported by the results shown in Table 3.

Scarified corneas of mice were inoculated with 1x10⁶ pfu of either HSV-1(F) or each of the recombinant viruses. The trigeminal ganglion were harvested 21 days post inoculation and processed for the detection of latent virus as described in Methods. As shown in Table 3, HSV-1(F) was detected in 94% of the trigeminal ganglia, while no latent virus was rescued from mice inoculated with the recombinant viruses. As a more direct approach, we analyzed trigeminal ganglia for latent virus following sublethal i.c. inoculations. As seen in Table 4, while latent virus was readily detected from the trigeminal ganglion of mice inoculated with HSV-1(F), we were again unable to recover latent virus from mice inoculated with either of the three recombinant viruses.

These results are particularly noteworthy since both HSV-1(F) and the recombinant viruses reach the trigeminal ganglion following i.c. inoculations. As shown in Table 5, acute virus can be detected through day 3 in the trigeminal ganglion of mice inoculated i.c. with either HSV-1(F) or any of the three recombinant viruses. Recent reports have shown that the use of either 50µM 5-azacytidine (AZC) (24) or 22mM dimethylsulfoxide (DMSO) (25) can increase the reactivation of HSV from latently infected trigeminal ganglia. Attempts to reactivate recombinant virus from trigeminal ganglia following sublethal i.c. inoculations with either 50µM AZC or 22 mM DMSO were also unsuccessful (Table 4). Therefore, the recombinant viruses either do not establish a latent infection in trigeminal ganglion or latent virus is not reactivable by the procedures used in this study.

Viral replication in brain

The studies on neuroinvasiveness indicated that the recombinant viruses may be capable of a limited replication in the corneas of mice, since infectious virus could be isolated 4 - 5 days post inoculation (Fig. 4). To determine if the recombinants were capable of replicating in other tissues of the mouse, we determined viral growth curves from the brains of BALB/c mice following sublethal i.c. inoculations. As shown in Figure 5, the recombinant viruses were capable of a minimal replication, maintaining viral titers of approximately 1x103 PFU/brain up to 4 days post inoculation.

Figure 5: Growth curves of HSV-1(F) and the recombinant viruses in Balb/C brains following intracerebral inoculation.