Interrelationship of Primary Virus Replication, Level of Latency, and Time to Reactivation in the Trigeminal Ganglia of Latently Infected Mice

Abstract

We sought to determine the possibility of an interrelationship between primary virus replication in the eye, the level of viral DNA in the trigeminal ganglia (TG) during latency, and the amount of virus reactivation following ocular herpes simplex virus type 1 (HSV-1) infection. Mice were infected with virulent (McKrae) or avirulent (KOS and RE) strains of HSV-1, and virus titers in the eyes and TG during primary infection, level of viral gB DNA in TG on day 28 postinfection (p.i.), and virus reactivation on day 28 p.i. as measured by explant reactivation were calculated. Our results suggest that the avirulent strains of HSV-1, even after corneal scarification, had lower virus titers in the eye, had less latency in the TG, and took a longer time to reactivate than virulent strains of HSV-1. The time to explant reactivation of avirulent strains of HSV-1 was similar to that of the virulent LAT((-)) McKrae-derived mutant. The viral dose with the McKrae strain of HSV-1 affected the level of viral DNA and time to explant reactivation. Overall, our results suggest that there is no absolute correlation between primary virus titer in the eye and TG and the level of viral DNA in latent TG and time to reactivation.

Importance: Very little is known regarding the interrelationship between primary virus replication in the eye, the level of latency in TG, and the time to reactivate in the mouse model. This study was designed to answer these questions. Our results point to the absence of any correlation between the level of primary virus replication and the level of viral DNA during latency, and neither was an indicator of how rapidly the virus reactivated following explant TG-induced reactivation.

FIG 1

Effect of LAT on the level of latency in different strains of mice. WT C57BL/6 and C57BL/6-CD8α^−/− mice were infected with LAT⁽⁺⁾ or LAT⁽⁻⁾ virus at 2 × 10⁵ PFU per eye. Twenty-eight days p.i., TG from infected mice were isolated and quantitative PCR was performed on each individual mouse TG. In each experiment, an estimated relative copy number of the HSV-1 gB for viral DNA was calculated using standard curves generated from pGem-gB1. Briefly, DNA template was serially diluted 10-fold such that 5 μl contained from 10³ to 10¹¹ copies of gB, and then it was subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH expression was used to normalize the relative expression of viral (gB) DNA in the TG. Each bar for WT mice is based on 40 TG from 2 separate experiments, while each bar for CD8α^−/− mice is based on 20 TG. Data are presented as means ± standard errors of the means (SEM).

FIG 2

Effect of LAT on induced reactivation in different strains of mice. WT C57BL/6 and C57BL/6-CD8α^−/− mice were infected with 2 × 10⁵ PFU per eye of LAT⁽⁺⁾ or LAT⁽⁻⁾ virus as described for Fig. 1. On day 28 p.i., TG from infected mice were isolated and each individual TG was incubated in 1.5 ml of tissue culture medium at 37°C. A 100-μl aliquot was removed from each culture daily for 15 days and used to infect RS cell monolayers. The RS cells were monitored daily for the appearance of CPE to determine the time of first appearance of reactivated virus from each TG. The results are plotted as the number of TG that reactivated daily. Numbers indicate the average time (±SEM) that the TG from each group first showed CPE. Each point represents the means ± SEM from 34 TG for WT [LAT⁽⁺⁾], 23 TG for WT [LAT⁽⁻⁾], 18 for CD8α^−/− [LAT⁽⁺⁾], and 18 for CD8α^−/− [LAT⁽⁻⁾] mice.

FIG 3

Virus titers in TG after ocular infection. WT C57BL/6 and CD8α^−/− mice were infected ocularly with 2 × 10⁵ PFU per eye of dLAT2903 [LAT⁽⁻⁾] or WT HSV-1 strain McKrae [LAT⁽⁺⁾] as described in Materials and Methods. Mice were euthanized on the indicated days. TG were removed and homogenized, and virus titers were determined as described in Materials and Methods. Each point represents the means ± SEM of titers from extracts of 10 TG.

FIG 4

Effect of dose of virus on the level of latency in TG of latently infected mice. WT C57BL/6 mice were ocularly infected with 1 × 10⁵, 2 × 10⁴, 1 × 10⁴, 2 × 10³, or 1 × 10³ PFU/eye of HSV-1 strain McKrae [LAT⁽⁺⁾] or dLAT2903 [LAT⁽⁻⁾] virus. On day 28 p.i., TG were harvested from the latently infected mice. Quantitative PCR was performed on each individual mouse TG. GAPDH expression was used to normalize the relative expression of viral (gB) DNA in the TG. Each point represents the means ± SEM from 20 TG. The Y-scale for 1 × 10⁴, 2 × 10³, and 1 × 10³ PFU is different from the Y-scale for 2 × 10⁵, 1 × 10⁵, and 2 × 10⁴ PFU. Data for 2 × 10⁵ PFU are repeated from the data shown in Fig. 1. (A) Latency in mice infected with 2 × 10⁵, 1 × 10⁵, or 2 × 10⁴ PFU/eye of LAT⁽⁺⁾ or LAT⁽⁻⁾ virus. (B) Latency in mice infected with 1 × 10⁴, 2 × 10³, or 1 × 10³ PFU/eye of LAT⁽⁺⁾ or LAT⁽⁻⁾ virus.

FIG 5

Effect of dose of HSV-1 on explant reactivation in TG of latently infected mice. WT C57BL/6 mice were ocularly infected with 1 × 10⁵, 2 × 10⁴, 1 × 10⁴, 2 × 10³, or 1 × 10³ PFU/eye of HSV-1 strain McKrae [LAT⁽⁺⁾] or dLAT2903 [LAT⁽⁻⁾] virus as described for Fig. 4. On day 28 p.i., TG from infected mice were harvested and explant reactivation was performed as described for Fig. 2. The presence of infectious virus in each culture was monitored daily for 15 days and used to infect RS cell monolayers. The RS cells were monitored daily for the appearance of CPE to determine the time of first appearance of reactivated virus from each TG. The results are plotted as the number of TG that reactivated daily. Numbers indicate the average time (±SEM) that the TG from each group first showed CPE. Each point represents the means ± SEM from 20 TG for each virus from two separate experiments.

FIG 6

Level of latency and duration of explant reactivation following ocular infection of mice with avirulent strains of HSV-1. Cornea from wt C57BL/6 mice were scarified before ocular infection and then were infected ocularly with 2 × 10⁵ PFU per eye of HSV-1 strains KOS and RE. On day 28 p.i., TG from infected mice were harvested for qPCR and explant reactivation. (A) gB DNA in latent TG. Quantitative PCR was performed on each mouse TG. GAPDH expression was used to normalize the relative expression of viral (gB) DNA in the TG. Each point represents the means ± SEM from 20 TG. (B) Explant reactivation in latent TG. Each individual TG from infected mice was incubated in 1.5 ml of tissue culture medium at 37°C, and the presence of infectious virus was monitored for 15 days as described for Fig. 2. For each virus, 20 TG from 10 mice were used. In KOS-infected mice, only 50% of latent TG were positive for the appearance of CPE, while in the RE strain 90% of TG showed CPE. Numbers indicate the average time (±SEM) that the TG from each group first showed CPE. (A) gB DNA; (B) explant reactivation.

FIG 7

Virus titers in the eyes of infected mice. WT C57BL/6 mice were ocularly infected with 2 × 10⁵ PFU per eye of HSV-1 strains McKrae [LAT⁽⁺⁾], dLAT2903 [LAT⁽⁻⁾], RE [LAT⁽⁺⁾], and KOS [LAT⁽⁺⁾] as described in Materials and Methods. The presence of infectious virus in the eyes of infected mice was monitored on days 3 and 5 p.i. by collecting tear films from 20 eyes for each virus. Each point represents the means ± SEM from 20 eyes.