Herpes simplex virus (HSV) types 1 and 2 reactivate from dissociated cultured dorsal root ganglia of latently infected mice. The neurons in culture were identified morphologically and by using the specific anti-neuronal monoclonal antibody A2B5. HSV antigen expression during reactivation was first seen in neurons on day 3 after dissociation, and infectious virus was released subsequently. Approximately 0.4~ of neurons released infectious virus. The presence of neutralizing antibody to HSV did not modify the reactivation process. After infection of mouse neuronal cultures in vitro with HSV, cytopathic effect and viral antigen expression appeared within 18 h predominantly in fibroblastic cells. It is well established that herpes simplex virus (HSV) can remain in latent form in sensory ganglia of experimental animals and man (Wildy et al., 1982). Previous studies have generally used ganglion explants to study virus release in vitro; the one study (Walz et al., 1976) which has claimed successful reactivation of HSV from dissociated dorsal root ganglia (DRG) of mice latently infected with virus has not been confirmed by others (Wildy et al., 1982). Recent evidence has suggested that neurons within DRG harbour latent HSV (McLennan & Darby, 1980). We have employed indirect immunofluorescence using A2B5 [a neuron-specific marker (Eisenbath et al., 1979; Walsh, 1980)], a polyspecific rabbit antiserum to HSV and a monoclonal antibody to HSV glycoprotein D (gD), to study reactivation in dissociated cell cultures prepared from DRG of mice latently infected with HSV types 1 and 2. As shown below, the latent HSV reactivates in such cultures as early as 3 days after dissociation. Initial viral antigen expression and virus reactivation, producing loss of processes and cell rounding occur in neurons. Three-week-old Pirbright or Biozzi (high antibody responder) strain mice were infected with wild-type HSV-2 or HSV-1 respectively. As previously described (A1-Saadi et al., 1983; Clements & Subak-Sharpe, 1983), 10 s p.f.u, of virus were inoculated into the right hind footpad of mice which were then kept for not less than 3 months until sacrificed. No mice showed any clinical evidence of active infection with HSV when sacrificed. Uninfected mice of the same strain were used as controls. The mice were killed with chloroform and the thoracic 12, 13, lumbar 1 to 6 and sacral 1, 2 DRG from each side were removed aseptically and washed in phosphate-buffered saline containing 25 p.g/ml gentamicin (PBS). The ganglia from two to five mice were pooled for each experiment, ganglia from the right side forming one pool and those from the left (contralateral to the site of inoculation) a second pool. Dissociated cell cultures of adult mouse DRG were prepared using modifications of previously described techniques (Kennedy et al., 1980). The pooled ganglia were gently teased apart with fine forceps and transferred to vials in which there was 2 ml of PBS containing 0.25% collagenase (Worthington) and incubated for 2-5 h at 37 °C. The ganglia were th...
SUMMARYHerpes simplex virus type 2 wild-type and 13 temperature-sensitive mutants have been examined for their ability to be recovered from latently infected Biozzi mice. After inoculation into the rear footpad, virus could be recovered from both the dorsal root ganglia (DRG) and the footpad (FP) at the site of inoculation (wt, ts 1, ts 2, ts 4, ts 6, ts 7 and ts 8), or from the DRG only (ts 11 and ts 13), or from FP only (ts 3, ts 9, ts 12). Two mutants (ts 5 and ts 10) have not been recovered from either site. Both DNApositive and DNA-negative mutants have been shown to be capable of establishing latent infection.Following a primary infection in man, herpes simplex virus (HSV) usually establishes latency, from which it may periodically reactivate and release virus; this can result in overt clinical disease. HSV genetic information persists in sensory ganglia both in man and in experimental animals, and after explantation of ganglia followed by culture in vitro the latent virus can be reactivated (Stevens & Cook, 1971;Stevens, 1975;Bastian et al., 1972;Barringer, 1975).HSV-1 temperature-sensitive (ts) mutants have been used to probe the mechanism of latency, and differences in ability to be recovered from latency have been described (Lofgren et al., 1977;Watson et al., 1980; Clements & Subak-Sharpe, 1983). In this paper we describe for the first time the ability of HSV-2 ts mutants to be recovered from the latent state in mouse dorsal root ganglia (DRG). In addition, we have been able to recover HSV-2 from another tissue, the footpad (FP) at the site of inoculation 3 months or more after infection.HSV-2 wild-type (wt) virus and a set of 13 ts mutants were used (Timbury, 1971 ;Halliburton & Timbury, 1973, 1976 to inoculate 3-to 4-week-old Biozzi high antibody responder strain mice of both sexes via the rear FP (10 s to 106 p.f.u.). No significant difference in response between males and females was found. It is relevant that the mouse body core temperature (38.5 °C) is non-permissive for the ts mutants used. The FP may be somewhat below the core temperature when the mice are active; however, when they are closely packed asleep in the nest, the FP temperature is probably at, or very close to, that of the core. No animal died or became paralysed following infection of Biozzi mice with the HSV-2 wt and ts mutants at the doses indicated in Table 1. Some mice showed slight FP swelling immediately after the initial inoculation, but no swelling or symptoms of active infection were noted subsequently.At least 3 months after primary infection (when the mice were asymptomatic), the animals were killed using chloroform. Nine ipsilateral (one sacral, six lumbar, two thoracic) and two contralateral DRG were rapidly dissected out and explanted under aseptic conditions. In addition, FP tissue from both the left and the right rear limbs was also explanted from each mouse and grown as organ cultures in vitro at 31 °C. In 16 cases out of a total of 199, the explanted FP became contaminated during culture and had to be discarded. ...
SUMMARYA subgenomic cDNA clone from human rhinovirus 14 , comprising the 5' non-coding region and the first 1182 nucleotides of the coding sequence, has been inserted into a vector under the control of the T7 promoter, and RNA was transcribed. Deletions in the 5' non-coding sequence modulated viral polyprotein synthesis significantly in a reticulocyte lysate system. Removal of the first 491 nucleotides had little effect, but deletion of a further 55 nucleotides (491 to 546) significantly increased the efficiency of the translation process. Further deletion to nucleotide 621 almost abolished translation, suggesting an essential role for the 546 to 621 nucleotide sequence. The efficiency of the translation process can also be influenced by the addition of ribosomal salt wash prepared from uninfected HeLa cells.
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