Herpes simplex virus: recurrent and nonrecurrent strains

A study of three Herpes Simplex strains with different frequencies of recurrent disease was done using the New Zealand white rabbit eye model. Each of the three strains, the McKrae strain (high frequency), the E-43 strain (low, frequency), and the CGA-3 (no recurrence) grew well in the rabbit corneal epithelium and produced overt recognizable disease for up to 5 days post-infection, thus minimizing differences in virus reactivation due to a lack of or insufficient ganglionic colonization. Asymptomatic shedding and spontaneous recurrences, as well as iontophoretically induced recurrences, were seen in the E-43 and McKrae strains, but not in the CGA-3-infected animals. The virus strain's optimum temperature was an important aspect of its reactivation process, as shown by the failure of the nonrecurrent CGA-3 to replicate at the host's core temperature (39 degrees C). The fact that these explants yielded infectious virus at 33 degrees C and not at 39 degrees C confirmed that the CGA-3 had colonized the ganglia, and its lack of recurrences or shedding suggests a temperature-dependency relationship. Our observations were further supported by the preferential growth at 39 degrees C of fresh clinical isolates obtained from HSV encephalitis and herpes labialis. Isolates from animals infected with the heterogeneous McKrae were classified as shedders (isolated in the absence of disease) and recurrent (isolated from a recurrent lesion). Both shedders and recurrent isolates were of a homologous nature and retained their phenotype when tested. From this study, we theorize that reactivation and disease may have different regulatory mechanisms. The type of recurrent disease (lesions, asymptomatic shedding, or none) is virus-dependent and frequency of disease may be regulated by host functions.     

Neutralizing Antibodies Inhibit Axonal Spread of Herpes Simplex Virus Type 1 to Epidermal Cells In Vitro

The ability of antibodies to interfere with anterograde transmission of herpes simplex virus (HSV) from neuronal axons to the epidermis was investigated in an in vitro model consisting of human fetal dorsal root ganglia innervating autologous skin explants in a dual-chamber tissue culture system. The number and size of viral cytopathic plaques in epidermal cells after axonal transmission from HSV type 1 (HSV-1)-infected dorsal root ganglionic neurons were significantly reduced by addition to the outer chamber of neutralizing polyclonal human sera to HSV-1, of a human recombinant monoclonal group Ib antibody to glycoprotein D (gD), and of rabbit sera to HSV-1 gB and gD but not by rabbit anti-gE or anti-gG. A similar pattern of inhibition of direct infection of epidermal cells by these antibodies was observed. High concentrations of the monoclonal anti-gD reduced transmission by 90%. Rabbit anti-gB was not taken up into neurons, and human anti-gD did not influence spread of HSV in the dorsal root ganglia or axonal transport of HSV antigens when applied to individual dissociated neurons. These results suggest that anti-gD and -gB antibodies interfere with axonal spread of HSV-1, possibly by neutralizing HSV during transmission across an intercellular gap between axonal termini and epidermal cells, and thus contribute to control of HSV spread and shedding. Therefore, selected human monoclonal antibodies to protective epitopes might even be effective in preventing epidermis-to-neuron transmission during primary HSV infection, especially neonatal infection.     

Reinfection of guinea pigs with herpes simplex virus type 2: failure to establish a second latent ganglionic infection

Guinea pigs were infected with herpes simplex virus type 2 (HSV 2) and at various times thereafter reinfected at a different site with the same or a different strain of HSV 2. Viruses were isolated from infected tissues either from the site of primary or secondary infection or from regional ganglia. Viral isolates were characterised by DNA fingerprinting using restriction endonucleases capable of differentiating between the virus strains used. The second infection resulted in a second site of peripheral persistence of the virus, but did not establish a second site of ganglionic infection.     

Latent Herpes Simplex Virus from Trigeminal Ganglia of Rabbits with Recurrent Eye Infection

LATENTLY infected sensory ganglia have been thought to be the source of virus for various clinical manifestations of recurrent herpetic disease in man^1,2. In direct support of this concept, we recently showed that herpes simplex virus can induce a latent infection in the spinal ganglia of mice³. This murine infection has not, however, been shown to be accompanied by recurrent disease. Recurrent herpetic eye infection can be produced in the rabbit⁴. If sensory ganglia are involved in recurrent disease, then trigeminal ganglia from rabbits undergoing such recurrent infection would be expected to harbour latent virus. We now report that herpes simplex virus does indeed induce latent infection in trigeminal ganglia of rabbits presenting recurrent eye infection. As in the experiments with mice, infectious virus could not be recovered directly; it was only found when ganglia were established as organ cultures in vitro.     

Eradication of herpes simplex virus persistence in rat trigeminal ganglia by retrograde axoplasmic transport.

Potential use of retrograde axoplasmic flow to eradicate virus persistence in ganglionic cells was studied in a new herpes simplex virus (HSV) persistence model in rat trigeminal ganglia. After injection of the F strain of HSV type 1 into the mental nerve, viral antigens were detectable in the ganglia by the immunofluorescence and peroxidase methods between postinoculation (p.i.) days 3 and 6 but not thereafter. None of 82 inoculated rats showed signs of acute illness, and some survived for more than 502 days without symptoms. By cocultivation of ganglion tissues with Vero cells, the virus was isolated from 42 of 49 ganglia (85.7%) between 15 and 386 days (p.i.). HSV DNA was solely localized in the nucleus of neurons by immunoperoxidase staining of paraffin sections with a biotinylated HSV DNA probe, and the presence of HSV DNA-positive cells was confirmed in four of four ganglia on p.i. day 6 and in five of six on p.i. day 502. The efficacy of axoplasmic flow in drug delivery to ganglionic cells was investigated by injection of doxorubicin (ADM) into the nerve once used for virus inoculation. As early as 19 h after injection, strong ADM-specific autofluorescence was seen in the nuclei of neurons parental to the mental nerve and in those of adjacent Schwann cells, and the death of ADM-positive cells subsequently ensued. A single injection of ADM reduced the virus isolation rate from 31/37 (84%) to 3/37 (8%).     

Quantitative polymerase chain reaction analysis of herpes simplex virus DNA in ganglia of mice infected with replication-incompetent mutants.

To study the roles of viral genes in the establishment and maintenance of herpes simplex virus (HSV) latency, we have developed a polymerase chain reaction assay that is both quantitative and sensitive. Using this assay, we analyzed the levels of viral DNA in trigeminal ganglia of mice inoculated corneally with HSV mutants that are defective for virus replication at one or more sites in mice and for reactivation upon ganglionic explant. Ganglia from mice infected with thymidine kinase-negative mutants, which replicate at the site of inoculation and establish latency but do not replicate acutely in ganglia or reactivate upon explant, contained a range of levels of HSV DNA that overlapped with the range found in ganglia latently infected with wild-type virus. On average, these mutant-infected ganglia contained one copy of HSV DNA per 100 cell equivalents (ca. 10(4) molecules), which was 50-fold less than the average for wild-type virus. Ganglia from mice infected with a ribonucleotide reductase deletion mutant, which is defective for acute replication and reactivation upon ganglionic explant, also contained on average one copy of HSV DNA per 100 cell equivalents. We also detected substantial numbers of HSV DNA molecules (up to ca. 10(3] in ganglia of mice infected with an ICP4 deletion mutant and other replication-negative mutants that are severely impaired for viral DNA replication and gene expression. These results raise the possibility that such mutants can establish latency, which could have important implications for mechanisms of latency and for vaccine and antiviral drug development.     

Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice

During lytic infection, the virion host shutoff (vhs) protein of herpes simplex virus (HSV) mediates the rapid degradation of RNA and shutoff of host protein synthesis. In mice, HSV type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. HSV-2 has significantly higher vhs activity than HSV-1, eliciting a faster and more complete shutoff. To examine further the role of vhs activity in pathogenesis, we generated an intertypic recombinant virus (KOSV2) in which the vhs open reading frame of HSV-1 strain KOS was replaced with that of HSV-2 strain 333. KOSV2 and a marker-rescued virus, KOSV2R, were characterized in cell culture and tested in an in vivo mouse eye model of latency and pathogenesis. The RNA degradation kinetics of KOSV2 was identical to that of HSV-2 333, and both showed vhs activity significantly higher than that of KOS. This demonstrated that the fast vhs-mediated degradation phenotype of 333 had been conferred upon KOS. The growth of KOSV2 was comparable to that of KOS, 333, and KOSV2R in cell culture, murine corneas, and trigeminal ganglia and had a reactivation frequency similar to those of KOS and KOSV2R from explanted latently infected trigeminal ganglia. There was, however, significantly reduced blepharitis and viral replication within the periocular skin of KOSV2-infected mice compared to mice infected with either KOS or KOSV2R. Taken together, these data demonstrate that heightened vhs activity, in the context of HSV-1 infection, leads to increased viral clearance from the skin of mice and that the replication of virus in the skin is a determining factor for blepharitis. These data also suggest a role for vhs in modulating host responses to HSV infection.     

Trigeminal ganglion infection by thymidine kinase-negative mutants of herpes simplex virus after in vivo complementation.

Infection of trigeminal ganglion by herpes simplex virus (HSV) thymidine kinase-negative (TK-) mutants was investigated in mixed infection studies in mice. Mice were corneally inoculated with TK- HSV alone or with mixtures of TK- HSV-TK+ HSV. When inoculated alone, an arabinosylthymine-selected HSV type 1 TK- mutant and a HSV type 2 TK- deletion mutant infected mouse ocular tissues but rarely infected ganglion tissues. However, both TK- mutants readily infected ganglion tissues when they were inoculated in mixtures with TK+ HSV. By means of mixed infection studies, it was demonstrated that TK- HSV could readily establish acute and latent ganglion infections. It was thought that the frequent infection of trigeminal ganglion tissue by both TK- mutants after mixed TK(-)-TK+ HSV infection was the result of in vivo complementation. After mixed TK(-)-TK+ HSV infection and subsequent cultivation of ganglion explants in arabinosylthymine, results supported the conclusion that when TK- was present in ganglia it was in the same neurons that contained TK+ HSV.     

Rabbit model of rotavirus infection.

A new small animal model was developed to study parameters of rotavirus infections, including the active immune response. Seronegative New Zealand White rabbits (neonatal to 4 months old) were inoculated orally with cultivatable rabbit rotavirus strains Ala, C11, and R2 and with the heterologous simian strain SA11. The course of infection was evaluated by clinical findings, virus isolation (plaque assay and enzyme-linked immunosorbent assay), and serologic response. All four strains of virus were capable of infecting rabbits as determined by isolation of infectious virus from intestinal contents or fecal samples, by seroconversion, or by a combination of these methods. The responses differed depending on the virus strain used for inoculation. Rabbits remained susceptible to primary infection to at least 16 weeks of age (upper limit examined). Virus excretion in intestinal contents was detected from 6 h to 7 days postinoculation. RNA electropherotypes of inocula and viruses isolated from rabbits were the same in all samples tested. Transmission of Ala virus and R2 virus but not SA11 virus from inoculated animals to uninoculated controls also occurred. In a challenge experiment with Ala virus, 74- and 90-day-old rabbits were rechallenged with Ala 5 weeks after a primary infection with Ala. Virus was excreted in feces from 2 to 8 days after the primary infection but was not excreted after challenge. These results indicate that the rabbit provides an ideal model to investigate both the primary and secondary active immune responses to rotavirus infections and to evaluate candidate vaccines.     

Induction and inhibition of apoptosis by pseudorabies virus in the trigeminal ganglion during acute infection of swine

We examined the ability of pseudorabies virus (PRV) to induce and suppress apoptosis in the trigeminal ganglion during acute infection of its natural host. Eight pigs were intranasally inoculated with a virulent field strain of PRV, and at various early times after inoculation, the trigeminal ganglia were assessed histologically. PRV-infected cells were detected by use of immunohistochemistry and in situ hybridization, and apoptosis was identified by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. Light and electron microscopy was also used for morphological studies. Apoptosis was readily detected among infiltrating immune cells that were located surrounding PRV-infected neurons. The majority of PRV-infected neurons did not show morphological or histochemical evidence of apoptosis, even including those neurons that were surrounded by numerous inflammatory cells and exhibited profound pathological changes. However, neuronal virus-induced apoptosis also occurred but at a sporadic low level. These findings suggest that PRV is able to block apoptosis of infected trigeminal ganglionic neurons during acute infection of swine. Furthermore, our results also suggest that apoptosis of infiltrating inflammatory cells may represent an important viral mechanism of immune evasion.     

The effects of aminoguanidine on primary and recurrent ocular herpes simplex virus infection

In primary ocular herpes simplex virus (HSV) infection, nitric oxide may function to control viral replication and herpetic stromal keratitis (HSK) lesions. Recurrent HSK, manifested as corneal opacity and neovascularization, is the potentially blinding sequel to primary infection. Here, we assess the effects of nitric oxide synthase inhibition on a mouse model of recurrent HSK. In preliminary primary infection experiments, NIH inbred mice treated with aminoguanidine, an inhibitor of inducible nitric oxide synthase (iNOS), experienced no changes in post-infection tear, brain, or ganglia virus titers, but encephalitis-related mortality was elevated. After UV-B stimulated viral reactivation, iNOS inhibition did not affect virus shedding or clinical disease. In contrast to primary HSK, there was no exacerbation of mortality in recurrent disease. Our findings indicate that nitric oxide can be neuroprotective without antiviral effects in primary HSK, and does not play a significant role in the pathogenesis of recurrent HSK. Compared with data from other mouse strains, this work suggests that there may be a genetic component to the importance of NO in controlling ocular HSV infection.     

Encephalitis resulting from reactivation of latent herpes simplex virus in mice.

Herpes simplex virus (HSV) encephalitis was produced in mice from reactivation of latent virus. Two experimental models were used: the trigeminal model after corneal inoculation of HSV, and the hypoglossal model after tongue inoculation of HSV. In the trigeminal model, cyclophosphamide treatment induced reactivation of latent virus in ganglia but not in central nervous system tissue. Spread of the reactivated virus from ganglia to brain occurred only in mice deficient in anti-HSV antibody. In the hypoglossal model, sectioning of the hypoglossal nerve provoked chromatolysis in the corresponding central nervous system motor neurons and occasionally reactivated latent HSV in the brains of mice. These results suggest that HSV encephalitis can result from the spread of reactivated virus from ganglia to brain and also from in situ reactivation in brain.