Herpes simplex virus-specific CD8+ T cells can clear established lytic infections from skin and nerves and can partially limit the early spread of virus after cutaneous inoculation

HSV infects skin or mucosal epithelium as well as entering the sensory nerves and ganglia. We have used TCR-transgenic T cells specific for the immunodominant class I-restricted determinant from HSV glycoprotein B (gB) combined with a flank zosteriform model of infection to examine the ability of CD8+ T cells to deal with infection. During the course of zosteriform disease, virus rapidly spreads from the primary inoculation site in the skin to sensory dorsal root ganglia and subsequently reappears in the distal flank. Virus begins to be cleared from all sites about 5 days after infection when gB-specific CD8+ T cells first appear within infected tissues. Although activated gB-specific effectors can partially limit virus egress from the skin, they do so only at the earliest times after infection and are ineffective at halting the progression of zosteriform disease once virus has left the inoculation site. In contrast, these same T cells can completely clear ongoing lytic replication if transferred into infected immunocompromised RAG-1-/- mice. Therefore, we propose that the role of CD8+ T cells during the normal course of disease is to clear replicating virus after infection is well established rather than limit the initial spread of HSV from the primary site of inoculation.     

A herpes simplex virus type 1 mutant containing a nontransinducing Vmw65 protein establishes latent infection in vivo in the absence of viral replication and reactivates efficiently from explanted trigeminal ganglia.

Vmw65, a herpes simplex virus type 1 (HSV-1) tegument protein, in association with cellular proteins, transactivates viral immediate early genes. In order to examine the role of Vmw65 during acute and latent infection in vivo, a mutant virus (in1814), containing a 12-base-pair insertion in the Vmw65 gene, which lacks the transactivating function of Vmw65 (C. I. Ace, T. A. McKee, J. M. Ryan, J. M. Cameron, and C. M. Preston, J. Virol. 63:2260-2269, 1989) was examined in mice. Following corneal inoculation, the parental virus (17+) and the revertant (1814R) replicated effectively in eyes and trigeminal ganglia with 30 to 60% mortality. At either equal PFU or equal particle numbers, in1814 did not replicate in trigeminal ganglia and none of the infected mice died. Although in1814 did not replicate following corneal inoculation, it established latent infection in trigeminal ganglia. HSV-1 in1814 reactivated at explant as efficiently and rapidly as did 17+ and 1814R. Even low amounts of inoculated in1814 (10(2) PFU) were sufficient to establish latent infection in some animals. Since infectious in1814 was not detected at any time in mouse trigeminal ganglia, in1814 provided a unique opportunity to determine how soon after primary infection latency begins. Latent in1814 infection was detected shortly after virus reached the sensory ganglia, between 24 to 48 h postinfection. Thus, though Vmw65 may be required for lytic infection in vivo, it is dispensable for the establishment of and reactivation from latent infection. These data support the hypotheses that the latent and lytic pathways of HSV-1 are distinct and that latency is established soon after infection without a requirement for viral replication. However, the levels of Vmw65 reaching neuronal nuclei may be a critical determinant of whether HSV-1 forms a lytic or latent infection.     

Direct entry of rabies virus into the central nervous system without prior local replication.

Rabies virus pathogenesis was studied in a mouse model by inoculation of the masseter muscle. At different intervals, the masseter muscle, trigeminal ganglia, and brain were analyzed for virus-specific RNA with a polymerase chain reaction assay, which revealed that as early as 18 h postinfection (p.i.), virus-specific RNA was present in the trigeminal ganglia, and at 24 h p.i., viral RNA was identified in the brain stem. Analysis of the masseter muscle demonstrated virus at 1 h p.i. but no virus-specific RNA between 6 and 30 h p.i., indicating that virus invaded the nerve ending directly, without prior replication in the muscle. At 36 h p.i., viral RNA was detected again in the masseter muscle. Selective amplification of plus- and minus-strand RNA isolated from the masseter muscle at 96 h p.i. revealed that the majority of the rabies virus-specific RNA was in the positive sense, suggesting virus replication in muscle tissue during late stages of infection.     

Neutrophil-mediated suppression of virus replication after herpes simplex virus type 1 infection of the murine cornea.

Herpes simplex virus type 1 (HSV-1) infection of the murine cornea induces the rapid infiltration of neutrophils. We investigated whether these cells could influence virus replication. BALB/c mice treated with monoclonal antibody (MAb) RB6-8C5 experienced a profound depletion of neutrophils in the bloodstream, spleen, and cornea. In these animals, virus titers in the eye were significantly higher than those in the immunoglobulin G-treated controls at 3 days postinfection. By day 9, virus was no longer detectable in the controls, whereas titers of 10(3) to 10(6) PFU were still present in the neutrophil-depleted hosts. Furthermore, virus spread more readily to the skin and brains of MAb RB6-8C5-treated animals, rendering them significantly more susceptible to HSV-1-induced blepharitis and encephalitis. Only 25% of the treated animals survived, whereas all of the controls lived. Although MAb RB6-8C5 treatment did not alter the CD4+ T-cell, B-cell, natural killer cell, or macrophage populations, the CD8+ T-cell population was partially reduced. Therefore, the experiments were repeated in severe combined immunodeficiency mice, which lack CD8+ T cells. Again virus growth was found to be significantly elevated in the eyes, trigeminal ganglia, and brains of the MAb RB6-8C5-treated hosts. These results strongly indicate that in both immunocompetent and immunodeficient mice, neutrophils play a significant role in helping to control the replication and spread of HSV-1 after corneal infection.     

Role of antibody in primary and recurrent herpes simplex virus infection.

When herpes simplex virus was inoculated into the flank of a BALB/c mouse by scarification, the local replication of virus was followed by the establishment of an acute ganglionic infection. The subsequent centrifugal spread of this virus along nerves to the skin of the whole dermatome led to the development of a bandlike "zosteriform" rash. This represents a highly reproducible system in which virus travels through the nervous system synchronously in large numbers of animals. The transection of peripheral nerves at various times after infection showed that the virus had completed the round trip 60 h after inoculation into the upper flank and was detectable as infectious virus by 74 h postinfection. After the administration of virus, neutralizing but not nonneutralizing antibodies prevented the development of the zosteriform rash. The target epitopes of the protective antibodies were not confined to a single glycoprotein. Neutralizing antibody was effective even when given up to 60 h postinfection and was protective even when administered after sensory neurotomy at this time. Antibody was therefore able to prevent clinically and virologically detectable infection of the skin, presumably by acting peripherally on virus emerging from nerve endings. A quantitative estimate of the action of one of the neutralizing monoclonal antibody preparations, AP7, showed that high titers (several times higher than those normally found in immune mice) were needed to prevent this type of infection. These results are discussed in relation to antibody prophylaxis.     

Herpes Simplex Virus 1 Targets the Murine Olfactory Neuroepithelium for Host Entry

Herpes simplex virus 1 (HSV-1) is a ubiquitous and important human pathogen. It is known to persist in trigeminal ganglia (TG), but how it reaches this site has been difficult to determine, as viral transmission is sporadic, pathogenesis is complicated, and early infection is largely asymptomatic. We used mice to compare the most likely natural HSV-1 host entry routes: oral and nasal. Intranasal infection was 100-fold more efficient than oral and targeted predominantly the olfactory neuroepithelium. Live imaging of HSV-1-expressed luciferase showed infection progressing from the nose to the TG and then reemerging in the facial skin. The brain remained largely luciferase negative throughout. Infected cell tagging by viral Cre recombinase expression in floxed reporter gene mice showed nasal virus routinely reaching the TG and only rarely reaching the olfactory bulbs. Thus, HSV-1 spread from the olfactory neuroepithelium to the TG and reemerged peripherally without causing significant neurological disease. This recapitulation of typical clinical infection suggests that HSV-1 might sometimes also enter humans via the respiratory tract.     

In vivo replication of an ICP34.5 second-site suppressor mutant following corneal infection correlates with in vitro regulation of eIF2 alpha phosphorylation

In animal models of herpes simplex virus type 1 (HSV-1) infection, ICP34.5-null viruses are avirulent and also fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. We show here that the inability of ICP34.5 mutants to grow in vitro is due specifically to the accumulation of phosphorylated eIF2 alpha. Mutations suppressing the in vitro phenotype of ICP34.5-null mutants have been described which map to the unique short region of the HSV-1 genome, resulting in dysregulated expression of the US11 gene. Despite the inability of the suppressor mutation to suppress the avirulent phenotype of the ICP34.5-null parental virus following intracranial inoculation, the suppressor mutation enhanced virus growth in the cornea, trigeminal ganglia, and periocular skin following corneal infection compared to that with the ICP34.5-null virus. The phosphorylation state of eIF2 alpha following in vitro infection with the suppressor virus was examined to determine if in vivo differences could be attributed to differential regulation of eIF2 alpha phosphorylation. The suppressor virus prevented accumulation of phosphorylated eIF2 alpha, while the wild-type virus substantially reduced eIF2 alpha phosphorylation levels. These data suggest that US11 functions as a PKR antagonist in vivo, although its activity may be modulated by tissue-specific differences in translation regulation.     

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.     

Inflammatory infiltration of the trigeminal ganglion after herpes simplex virus type 1 corneal infection.

Following herpes simplex virus type 1 (HSV-1) infection of the cornea, the virus is transmitted to the trigeminal ganglion, where a brief period of virus replication is followed by establishment of a latent infection in neurons. A possible role of the immune system in regulating virus replication and maintaining latency in the sensory neurons has been suggested. We have investigated the phenotype and cytokine pattern of cells that infiltrate the A/J mouse trigeminal ganglion at various times after HSV-1 corneal infection. HSV antigen expression in the trigeminal ganglion (indicative of the viral lytic cycle) increased until day 3 postinfection (p.i.) and then diminished to undetectable levels by day 7 p.i. The period of declining HSV antigen expression. was associated with a marked increase in Mac-1+ cells. These cells did not appear to coexpress the F4/80+ (macrophage) or the CD8+ (T cell) markers, and none showed polymorphonuclear leukocyte morphology, suggesting a possible early infiltration of natural killer cells. There was also a significant increase in the trigeminal ganglion of cells expressing the gamma delta T-cell receptor, and these cells were found almost exclusively in very close association with neurons. This period was also characterized by a rapid and equivalent increase in cells expressing gamma interferon and interleukin-4. The density of the inflammatory infiltrate in the trigeminal ganglion increased until days 12 to 21 p.i., when it was predominated by CD8+, Mac-1+, and tumor necrosis factor-expressing cells, which surrounded many neurons. By day 92 p.i., the inflammatory infiltrate diminished but was heaviest in mice with active periocular skin disease. Our data are consistent with the notion that gamma interferon produced by natural killer cells and/or gamma delta T cells may play an important role in limiting HSV-1 replication in the trigeminal ganglion during the acute stage of infection. In addition, tumor necrosis factor produced by CD8+ T cells and macrophages may function to maintain the virus in a latent state.     

Herpes simplex virus type 1 glycoprotein E domains involved in virus spread and disease

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) functions as an immunoglobulin G (IgG) Fc binding protein and is involved in virus spread. Previously we studied a gE mutant virus that was impaired for IgG Fc binding but intact for spread and another that was normal for both activities. To further evaluate the role of gE in spread, two additional mutant viruses were constructed by introducing linker insertion mutations either outside the IgG Fc binding domain at gE position 210 or within the IgG Fc binding domain at position 380. Both mutant viruses were impaired for spread in epidermal cells in vitro; however, the 380 mutant virus was significantly more impaired and was as defective as gE null virus. gE mutant viruses were inoculated into the murine flank to measure epidermal disease at the inoculation site, travel of virus to dorsal root ganglia, and spread of virus from ganglia back to skin to produce zosteriform lesions. Disease at the inoculation and zosteriform sites was reduced for both mutant viruses, but more so for the 380 mutant virus. Moreover, the 380 mutant virus was highly impaired in its ability to reach the ganglia, as demonstrated by virus culture and real-time quantitative PCR. The results indicate that the domain surrounding amino acid 380 is important for both spread and IgG Fc binding and suggest that this domain is a potential target for antiviral therapy or vaccines.     

Oligoadenylate synthetase/protein kinase R pathways and alphabeta TCR+ T cells are required for adenovirus vector: IFN-gamma inhibition of herpes simplex virus-1 in cornea

An adenoviral (Ad) vector containing the murine IFN-gamma transgene (Ad:IFN-gamma) was evaluated for its capacity to inhibit HSV-1. To measure effectiveness, viral titers were analyzed in cornea and trigeminal ganglia (TG) during acute ocular HSV-1 infection. Ad:IFN-gamma potently suppressed HSV-1 replication in a dose-dependent fashion, requiring IFN-gamma receptor. Moreover, Ad:IFN-gamma was effective when delivered -72 and -24 h before infection as well as 24 h postinfection. Associated with antiviral opposition, TG from Ad:IFN-gamma-transduced mice harbored fewer T cells. Also related to T cell involvement, Ad:IFN-gamma was effective but attenuated in TG from alphabeta TCR-deficient mice. In corneas, alphabeta TCR(+) T cells were obligatory for protection against viral multiplication. Type I IFN involvement amid antiviral efficacy of Ad:IFN-gamma was further investigated because types I and II IFN pathways have synergistic anti-HSV-1 activity. Ad:IFN-gamma inhibited viral reproduction in corneas and TG from alphabeta IFNR-deficient (CD118(-/-)) mice, although viral titers were 2- to 3-fold higher in cornea and TG compared with wild-type mice. The absence of IFN-stimulated antiviral proteins, 2'-5' oligoadenylate synthetase/RNase L, and dsRNA-dependent protein kinase R completely eliminated the antiviral effectiveness of Ad:IFN-gamma. Collectively, the results demonstrate the following: 1) nonexistence of type I IFN receptor does not abolish defense of Ad:IFN-gamma against HSV-1; 2) antiviral pathways oligoadenylate synthetase-RNase L and protein kinase R are mandatory; and 3) alphabeta TCR(+) T cells are compulsory for Ad:IFN-gamma effectiveness against HSV-1 in cornea but not in TG.     

Influenza A Virus Encoding Secreted Gaussia Luciferase as Useful Tool to Analyze Viral Replication and Its Inhibition by Antiviral Compounds and Cellular Proteins

Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.     

Mice with mutations in Fas and Fas ligand demonstrate increased herpetic stromal keratitis following corneal infection with HSV-1

HSV-1 infection of the cornea leads to a potentially blinding immunoinflammatory lesion of the cornea, termed herpetic stromal keratitis. It has also been shown that one of the factors limiting inflammation of the cornea is the presence of Fas ligand (FasL) on corneal epithelium and endothelium. In this study, the role played by FasL expression in the cornea following acute infection with HSV-1 was determined. Both BALB/c and C57BL/6 (B6) mice with HSV-1 infection were compared with their lpr and gld counterparts. Results indicated that mice bearing mutations in the Fas Ag (lpr) displayed the most severe disease, whereas the FasL-defective gld mouse displayed an intermediate phenotype. It was further demonstrated that increased disease was due to lack of Fas expression on bone marrow-derived cells. Of interest, although virus persisted slightly longer in the corneas of mice bearing lpr and gld mutations, the persistence of infectious virus in the trigeminal ganglia was the same for all strains infected. Further, B6 mice bearing lpr and gld mutations were also more resistant to virus-induced mortality than were wild-type B6 mice. Thus, neither disease nor mortality correlated with viral replication in these mice. Collectively, the findings indicate that the presence of FasL on the cornea restricts the entry of Fas(+) bone marrow-derived inflammatory cells and thus reduces the severity of HSK.