Explant Reactivation Research Articles

Assessment of Two Novel Live-Attenuated Vaccine Candidates for Herpes Simplex Virus 2 (HSV-2) in Guinea Pigs.

Treatment to ameliorate the symptoms of infection with herpes simplex virus 2 (HSV-2) and to suppress reactivation has been available for decades. However, a safe and effective preventative or therapeutic vaccine has eluded development. Two novel live-attenuated HSV-2 vaccine candidates (RVx201 and RVx202) have been tested preclinically for safety. Hartley guinea pigs were inoculated vaginally (n = 3) or intradermally (n = 16) with either vaccine candidate (2 × 107 PFU) and observed for disease for 28 days. All animals survived to study end without developing HSV-2-associated disease. Neither vaccine candidate established latency in dorsal root or sacral sympathetic ganglia, as determined by viral DNA quantification, LAT expression, or explant reactivation. Infectious virus was shed in vaginal secretions for three days following vaginal inoculation with RVx202, but not RVx201, although active or latent HSV-2 was not detected at study end. In contrast, guinea pigs inoculated with wild-type HSV-2 MS (2 × 105 PFU) vaginally (n = 5) or intradermally (n = 16) developed acute disease, neurological signs, shed virus in vaginal secretions, experienced periodic recurrences throughout the study period, and had latent HSV-2 in their dorsal root and sacral sympathetic ganglia at study end. Both vaccine candidates generated neutralizing antibody. Taken together, these findings suggest that these novel vaccine candidates are safe in guinea pigs and should be tested for efficacy as preventative and/or therapeutic anti-HSV-2 vaccines.

Heart Dysfunction Following Long-Term Murine Cytomegalovirus Infection: Fibrosis, Hypertrophy, and Tachycardia.

Human cytomegalovirus (HCMV) is associated with increased risk of chronic diseases of the heart and vasculature, including myocarditis, atherosclerosis, and transplant vasculopathy. To investigate CMV infection of the heart, murine cytomegalovirus (MCMV) was used to evaluate both acute and latent infection and the subsequent phenotypic and functional consequences of infection. Female BALB/c mice were intraperitoneally (i.p.) inoculated with 1 × 106 pfu of MCMV and evaluated at 14 and 50 days postinfection (dpi). At each time point, echocardiography was used to evaluate cardiac function and histology was conducted for phenotypic evaluation. MCMV replication in the heart was detected as early as 3 dpi and was no longer detectable at 14 dpi. Infected animals had significant cardiac pathology at 14 and 50 dpi when compared to uninfected controls. Histology revealed fibrosis of the heart as early as 14 dpi and the presence of white fibrous deposits on the surface of the heart. Functional evaluation showed significantly increased heart rate and muscle thickening in the latently infected animals when compared to the control animals. At 50 dpi, latent virus was measured by explant reactivation assay, demonstrating that MCMV establishes latency and is capable of reactivation from the heart, similar to other tissues such as spleen and salivary glands. Collectively, these studies illustrate that MCMV infection results in phenotypic alterations within the heart as early as 14 dpi, which progress to functional abnormalities during latency. These findings are similar to sinus tachycardia and hypertrophy of the heart muscle observed in cases of HCMV-induced acute myocarditis.

An M2 Rather than a TH2 Response Contributes to Better Protection against Latency Reactivation following Ocular Infection of Naive Mice with a Recombinant Herpes Simplex Virus 1 Expressing Murine Interleukin-4.

We found previously that altering macrophage polarization toward M2 responses by injection of colony-stimulating factor 1 (CSF-1) was more effective in reducing both primary and latent infections in mice ocularly infected with herpes simplex virus 1 (HSV-1) than M1 polarization by gamma interferon (IFN-γ) injection. Cytokines can coordinately regulate macrophage and T helper (TH) responses, with interleukin-4 (IL-4) inducing type 2 TH (TH2) as well as M2 responses and IFN-γ inducing TH1 as well as M1 responses. We have now differentiated the contributions of these immune compartments to protection against latency reactivation and corneal scarring by comparing the effects of infection with recombinant HSV-1 in which the latency-associated transcript (LAT) gene was replaced with either the IL-4 (HSV-IL-4) or IFN-γ (HSV-IFN-γ) gene using infection with the parental (LAT-negative) virus as a control. Analysis of peritoneal macrophages in vitro established that the replacement of LAT with the IL-4 or IFN-γ gene did not affect virus infectivity and promoted polarization appropriately. Protection against corneal scarring was significantly higher in mice ocularly infected with HSV-IL-4 than in those infected with HSV-IFN-γ or parental virus. Levels of primary virus replication in the eyes and trigeminal ganglia (TG) were similar in the three groups of mice, but the numbers of gC+ cells were lower on day 5 postinfection in the eyes of HSV-IL-4-infected mice than in those infected with HSV-IFN-γ or parental virus. Latency and explant reactivation were lower in both HSV-IL-4- and HSV-IFN-γ-infected mice than in those infected with parental virus, with the lowest level of latency being associated with HSV-IL-4 infection. Higher latency correlated with higher levels of CD8, PD-1, and IFN-γ mRNA, while reduced latency and T-cell exhaustion correlated with lower gC+ expression in the TG. Depletion of macrophages increased the levels of latency in all ocularly infected mice compared with their undepleted counterparts, with macrophage depletion increasing latency in the HSV-IL-4 group greater than 3,000-fold. Our results suggest that shifting the innate macrophage immune responses toward M2, rather than M1, responses in HSV-1 infection would improve protection against establishment of latency, reactivation, and eye disease.IMPORTANCE Ocular HSV-1 infections are among the most frequent serious viral eye infections in the United States and a major cause of virus-induced blindness. As establishment of a latent infection in the trigeminal ganglia results in recurrent infection and is associated with corneal scarring, prevention of latency reactivation is a major therapeutic goal. It is well established that absence of latency-associated transcripts (LATs) reduces latency reactivation. Here we demonstrate that recombinant HSV-1 expressing IL-4 (an inducer of TH2/M2 responses) or IFN-γ (an inducer of TH1/M1 responses) in place of LAT further reduced latency, with HSV-IL-4 showing the highest overall protective efficacy. In naive mice, this higher protective efficacy was mediated by innate rather than adaptive immune responses. Although both M1 and M2 macrophage responses were protective, shifting macrophages toward an M2 response through expression of IL-4 was more effective in curtailing ocular HSV-1 latency reactivation.

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

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. 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.

CD8+ T Cells Play a Bystander Role in Mice Latently Infected with Herpes Simplex Virus 1.

Based on an explant reactivation model, it has been proposed that CD8(+) T cells maintain latency in trigeminal ganglia (TG) of mice latently infected with herpes simplex virus 1 (HSV-1) [T. Liu, K. M. Khanna, X. Chen, D. J. Fink, and R. L. Hendricks, J Exp Med 191:1459-1466, 2000, doi:10.1084/jem.191.9.1459; K. M. Khanna, R. H. Bonneau, P. R. Kinchington, and R. L. Hendricks, Immunity 18:593-603, 2003, doi:10.1016/S1074-7613(03)00112-2]. In those studies, BALB/c mice were ocularly infected with an avirulent HSV-1 strain (RE) after corneal scarification. However, in our studies, we typically infect mice with a virulent HSV-1 strain (McKrae) that does not require corneal scarification. Using a combination of knockout mice, adoptive transfers, and depletion studies, we recently found that CD8α(+) dendritic cells (DCs) contribute to HSV-1 latency and reactivation in TG of ocularly infected mice (K. R. Mott, S. J. Allen, M. Zandian, B. Konda, B. G. Sharifi, C. Jones, S. L. Wechsler, T. Town, and H. Ghiasi, PLoS One 9:e93444, 2014, doi:10.1371/journal.pone.0093444). This suggested that CD8(+) T cells might not be the major regulators of HSV-1 latency in the mouse TG. To investigate this iconoclastic possibility, we used a blocking CD8 antibody and CD8(+) T cells in reactivated TG explants from mice latently infected with (i) the avirulent HSV-1 strain RE following corneal scarification or (ii) the virulent HSV-1 strain McKrae without corneal scarification. Independently of the strain or approach, our results show that CD8α(+) DCs, not CD8(+) T cells, drive latency and reactivation. In addition, adoptive transfer of CD8(+) T cells from wild-type (wt) mice to CD8α(-/-) mice did not restore latency to the level for wt mice or wt virus. In the presence of latency-associated transcript (LAT((+)); wt virus), CD8(+) T cells seem to play a bystander role in the TG. These bystander T cells highly express PD-1, most likely due to the presence of CD8α(+) DCs. Collectively, these results support the notion that CD8(+) T cells do not play a major role in maintaining HSV-1 latency and reactivation. This study addresses a fundamentally important and widely debated issue in the field of HSV latency-reactivation. In this article, we directly compare the effects of anti-CD8 antibody, CD8(+) T cells, LAT, and CD8α(+) DCs in blocking explant reactivation in TG of mice latently infected with avirulent or virulent HSV-1. Our data suggest that CD8(+) T cells are not responsible for an increase or maintenance of latency in ocularly infected mice. However, they seem to play a bystander role that correlates with the presence of LAT, higher subclinical reactivation levels, and higher PD-1 expression levels.

Mutations within the pathogenic region of herpes simplex virus 1 gK signal sequences alter cell surface expression and neurovirulence.

To investigate the role of the signal sequences of herpes simplex virus 1 (HSV-1) gK on virus replication and viral pathogenesis, we constructed recombinant viruses with or without mutations within the signal sequences of gK. These recombinant viruses expressed two additional copies of the mutated (MgK) or native (NgK) form of the gK gene in place of the latency-associated transcript with a myc epitope tag to facilitate detection at their 3' ends. The replication of MgK virus was similar to that of NgK both in vitro and in vivo, as well as in the trigeminal ganglia (TG) of latently infected mice. The levels of gB and gK transcripts in the corneas, TG, and brains of infected mice on days 3 and 5 postinfection were markedly virus and time dependent, as well as tissue specific. Mutation in the signal sequence of gK in MgK virus blocked cell surface expression of gK-myc in rabbit skin cells, increased 50% lethal dose, and decreased corneal scarring in ocularly infected mice compared to the NgK or revertant (RgK) virus. MgK and NgK viruses, and not the RgK virus, showed a reduced extent of explant reactivation at the lower dose of ocular infection but not at the higher dose. However, the time of reactivation was not affected by overexpression of the different forms of gK. Taken together, these results strongly suggest that the 8mer peptide (ITAYGLVL) within the signal sequence of gK promotes cell surface expression of gK in infected cells and ocular pathogenesis in infected mice. In this study, we show for the first time that mutations within the signal sequence of gK blocked cell surface expression of inserted recombinant gK in vitro. Furthermore, this blockage in cell surface expression was correlated with higher 50% lethal dose and less corneal scarring in vivo. Thus, these studies point to a key role for the 8mer within the signal sequence of gK in HSV-1-induced pathogenicity.

Factors affecting herpes simplex virus reactivation from the explanted mouse brain

The majority of encephalitis induced by herpes simplex virus type I (HSV-1) is due to viral reactivation from latency, but few studies have investigated the factors influencing viral reactivation in the brain due to the lack of a sensitive assay. We have established an ex vivo explant assay, which induced efficient viral reactivation in the dissociated mouse brain. Applying this assay, we investigated the infection of four HSV-1 strains with varying degrees of neurovirulence in three mouse strains with different levels of susceptibility to HSV-1 infection. We found that virulent HSV-1 strains and susceptible mouse strains exhibited prolonged viral growth during acute infection, increased latent viral genomes, and efficient explant reactivation in the brain stem. Collectively, both viral neurovirulence and host susceptibility positively correlate with HSV-1 reactivation from the explanted mouse brain.

Recombinant Herpes Simplex Virus Type 1 (HSV-1) Codelivering Interleukin-12p35 as a Molecular Adjuvant Enhances the Protective Immune Response against Ocular HSV-1 Challenge

An important aspect of ocular herpes simplex virus type 1 (HSV-1) vaccine development is identification of an appropriate adjuvant capable of significantly reducing both virus replication in the eye and explant reactivation in trigeminal ganglia. We showed recently that a recombinant HSV-1 vaccine expressing interleukin-4 (IL-4) is more efficacious against ocular HSV-1 challenge than recombinant viruses expressing IL-2 or gamma interferon (IFN-gamma) (Y. Osorio and H. Ghiasi, J. Virol. 77:5774-5783, 2003). We have now constructed and compared recombinant HSV-1 viruses expressing IL-12p35 or IL-12p40 molecule with IL-4-expressing HSV-1 recombinant virus. BALB/c mice were immunized intraperitoneally with IL-12p35-, IL-12p40-, IL-12p35+IL-12p40-, or IL-4-expressing recombinant HSV-1 viruses. Controls included mice immunized with parental virus and mice immunized with the avirulent strain KOS. The efficacy of each vaccine in protecting against ocular challenge with HSV-1 was assessed in terms of survival, eye disease, virus replication in the eye, and explant reactivation. Neutralizing antibody titers, T-cell responses, and expression of 32 cytokines and chemokines were also evaluated. Mice immunized with recombinant HSV-1 expressing IL-12p35 exhibited the lowest virus replication in the eye, the most rapid virus clearance, and the lowest level of explant reactivation. The higher efficacy against ocular virus replication and explant reactivation correlated with higher neutralizing antibody titers, cytotoxic-T-lymphocyte activities, and IFN-gamma expression in recombinant HSV-1 expressing IL-12p35 compared to other vaccines. Mice immunized with both IL-12p35 and IL-12p40 had lower neutralizing antibody responses than mice immunized with IL-12p35 alone. Our results confirm that recombinant virus vaccines expressing cytokine genes can enhance the overall protection against infection, with the IL-12p35 vaccine being the most efficacious of those tested. Collectively, the results support the potential use of IL-12p35 as a vaccine adjuvant, without the toxicity-associated concerns of IL-12.

Construction of a Herpes Simplex Virus Type 1 Mutant with Only a Three-Nucleotide Change in the Branchpoint Region of the Latency-Associated Transcript (LAT) and the Stability of Its Two-Kilobase LAT Intron

Previous studies using a eukaryotic expression system indicated that the unusual stability of the latency-associated transcript (LAT) intron was due to its nonconsensus branchpoint sequence (T.-T Wu, Y.-H. Su, T. M. Block, and J. M. Taylor, Virology, 243:140-149, 1998). The present study investigated the role of the branchpoint sequence in the stability of the intron expressed from the herpes simplex virus type 1 (HSV-1) genome and the role of LAT intron stability in the HSV-1 life cycle. A branchpoint mutant called Sy2 and the corresponding rescued viruses, SyRA and SyRB, were constructed. To preserve the coding sequence of the immediate early gene icp0, which overlaps with the branchpoint region of the 2-kb LAT, a 3-nucleotide mutation into the branchpoint region of the 2-kb LAT was introduced, resulting in a branchpoint that is 85% identical to the consensus intron branchpoint sequence of eukaryotic cells. As anticipated, there was a 90- to 96-fold reduction in 2-kb LAT accumulation following productive infection in tissue culture and latent infection in mice with Sy2, as determined by Northern blot analysis. These results clearly suggest that the accumulation of the 2-kb intron in tissue culture and in vivo is, at least in part, due to the nonconsensus branchpoint sequence of the LAT intron. Interestingly, a failure to accumulate LAT was associated with greater progeny production of Sy2 at a low multiplicity of infection (0.01) in tissue culture, but not in mice. However, the ability of mutant Sy2 to reactivate from trigeminal ganglia (TG) derived from latently infected mice was indistinguishable from that of wild-type virus, as assayed in the mouse TG explant reactivation system.

Replication and virulence of early protein 0 and long latency transcript deficient mutants of the Aujeszky′s disease (pseudorabies) virus

Early protein 0 (EP0)-deficient recombinant Aujeszky′s disease viruses, Ka-ep0lac and Ba-ep0lac derived from strains Kaplan and Bartha, respectively, were constructed to explore the impact of the mutation on replication, virulence and latency of the virus. Inactivation of the EP0 gene resulted in a mutation of long latency transcript (Cheung et al., 1991) that is located on the complementary DNA strand of EP0 and immediate early protein (IE)175 genes. In infection of immortalized porcine kidney cells, the growth rate and yield of both EP0 – mutant strains were significantly smaller than that of wild-type virus. Ka-ep0lac was found to be highly virulent, while Ba-ep0lac showed an attenuated phenotype in mice. PCR assay and immunohistochemistry showed that the Ba-ep0lac virus was able to establish latency in the mouse trigeminal ganglia. However, latent virus was not able to reactivate in explant reactivation assays. Accordingly, latent Ba-ep0lac has the potential to be exploited as vectors for the delivery of foreign genes to the nervous system.

Interferon gamma regulates acute and latent murine cytomegalovirus infection and chronic disease of the great vessels.

To define immune mechanisms that regulate chronic and latent herpesvirus infection, we analyzed the role of interferon gamma (IFN-gamma) during murine cytomegalovirus (MCMV) infection. Lethality studies demonstrated a net protective role for IFN-gamma, independent of IFN-alpha/beta, during acute MCMV infection. Mice lacking the IFN-gamma receptor (IFN-gammaR-/-) developed and maintained striking chronic aortic inflammation. Arteritis was associated with inclusion bodies and MCMV antigen in the aortic media. To understand how lack of IFN-gamma responses could lead to chronic vascular disease, we evaluated the role of IFN-gamma in MCMV latency. MCMV-infected IFN-gammaR-/- mice shed preformed infectious MCMV in spleen, peritoneal exudate cells, and salivary gland for up to 6 mo after infection, whereas the majority of congenic control animals cleared chronic productive infection. However, the IFN-gammaR was not required for establishment of latency. Using an in vitro explant reactivation model, we showed that IFN-gamma reversibly inhibited MCMV reactivation from latency. This was at least partly explained by IFN-gamma- mediated blockade of growth of low levels of MCMV in tissue explants. These in vivo and in vitro data suggest that IFN-gamma regulation of reactivation from latency contributes to control of chronic vascular disease caused by MCMV. These studies are the first to demonstrate that a component of the immune system (IFN-gamma) is necessary to regulate MCMV-associated elastic arteritis and latency in vivo and reactivation of a herpesvirus from latency in vitro. This provides a new model for analysis of the interrelationships among herpesvirus latency, the immune system, and chronic disease of the great vessels.

A 437-base-pair deletion at the beginning of the latency-associated transcript promoter significantly reduced adrenergically induced herpes simplex virus type 1 ocular reactivation in latently infected rabbits.

In this study we used a herpes simplex virus type 1 (HSV-1) deletion mutant to identify a segment of the genome necessary for epinephrine-induced reactivation in the rabbit eye model of herpetic recurrent disease. In HSV-1 latently infected neural tissue, the only abundant viral products are the latency-associated transcripts (LATs). At least one promoter of LAT has been identified, and mutations in the LAT domain have been used to investigate HSV-1 reactivation. We used an ocular rabbit model of epinephrine-induced HSV-1 reactivation to study the effects of deleting a 437-bp region beginning 796 bp upstream of the LAT CAP site. Specifically, the 437-bp deletion is located between genomic positions 118006 and 118443 of the parent 17Syn+, and the construct is designated 17 delta S/N. This region also controls a portion of the genome encoding two transcripts (1.1 and 1.8 kb) from the LAT domain. A rescuant, 17 delta S/N-Res, was constructed from 17 delta S/N. Following ocular infection, all three viruses produced similar acute dendritic lesions in rabbits. Five weeks after infection, rabbits received transcorneal iontophoresis of epinephrine. The parent, 17Syn+, and the rescuant, 17 delta S/N-Res, underwent a high frequency of HSV-1 ocular reactivation as determined by recovery of infectious virus in the tear film. Rabbits infected with 17 delta S/N had a significantly lower frequency of ocular reactivation. Analysis of the trigeminal ganglia from all three groups of latently infected rabbits revealed (i) similar amounts of HSV DNA (genomic equivalents), (ii) accumulation of 2.0- and 1.45-kb LATs, and (iii) explant reactivation at the same high frequency. Therefore, these studies indicate that the 437-bp deleted region in 17 delta S/N is essential for epinephrine-induced reactivation and could implicate the 1.1- and 1.8-kb transcripts in the mechanisms controlling HSV-1 reactivation.

Replication, establishment of latent infection, expression of the latency-associated transcripts and explant reactivation of herpes simplex virus type 1 gamma 34.5 mutants in a mouse eye model.

The herpes simplex virus type 1 (HSV-1) gamma 34.5 gene is located within a region that is transcriptionally active during latent HSV-1 infection. To determine whether the gamma 34.5 gene deletion affects latency-associated transcript (LAT) gene expression or latent HSV-1 infection, a gamma 34.5 gene deletion mutant, 1716, and a stop codon insertion mutant, 1771, were studied in the mouse eye model. Although the gamma 34.5 gene is not essential, 1716 and 1771 replicated poorly in mouse eyes and trigeminal ganglia (TG). When mice were inoculated with 1716, infectious virus was detected in eyes only on the first day post-infection (p.i.), and was not detected at any time point in TG. Following inoculation with 1771, a small amount of virus was detected in the eyes on days 2 and 4 p.i., and in the TG of one animal on day 2 p.i. Reactivation of virus from mice latently infected with 1716 (0/30 TG) and 1771 (1/20 TG) was extremely low compared with the parental strain, 17+, and appropriate rescuants (80 to 100% reactivation), even though latent 1716 DNA was detected by PCR in 50% of TG. These results differ from those obtained following footpad inoculation; in the footpad there was limited 1716 replication and reactivatable latent infection was established in some dorsal root ganglia. The data support the hypothesis that the role of gamma 34.5 may be tissue and/or cell type specific. The synthesis, processing, and stability of the 2.0 kb LAT during 1716 and 1771 replication was not affected by these mutations in the gamma 34.5 gene. However, during latent infection of 1716 in mice the LATs were not detectable in TG by Northern blot, and were present in reduced amounts (approximately 10-fold less) during 1771 latency. The LATs from 1716 were barely detectable in a few neurons by in situ hybridization. Therefore, the gamma 34.5 gene might (i) affect replication in the eye, and reduce the amount of virus available to establish latent infection, be directly involved in (ii) establishment of latency, and/or (iii) the reactivation process.

Two open reading frames (ORF1 and ORF2) within the 2.0-kilobase latency-associated transcript of herpes simplex virus type 1 are not essential for reactivation from latency.

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) are dispensable for establishment and maintenance of latent infection. However, the LATs have been implicated in reactivation of the virus from its latent state. Since the reported LAT deletion and/or insertion variants that are reactivation impaired contain deletions in the putative LAT promoter, it is not known which LAT sequences are involved in reactivation. To examine the role of the 2.0-kb LAT in the process of reactivation and the functional importance of the putative open reading frames (ORF1 and ORF2) contained within the 2.0-kb LAT, we have constructed an HSV-1 variant that contains a precise deletion and insertion within the LAT-specific DNA sequences using site-directed mutagenesis. The HSV-1 variant FS1001K contains an 1,186-bp deletion starting precisely from the 5' end of the 2.0-kb LAT and, for identification, a XbaI restriction endonuclease site insertion. The FS1001K genome contains no other deletions and/or insertions as analyzed by a variety of restriction endonucleases. The deletion in FS1001K removes the entire 556-bp intron within the 2.0-kb LAT, the first 229 nucleotides of ORF1, and the first 159 nucleotides of ORF2 without having an affect on the RL2 (ICP0) gene. Explant cocultivation reactivation assays indicated that this deletion had a minimal effect on reactivation of the variant FS1001K compared with the parental wild-type virus using a mouse eye model. As expected, Northern (RNA) blot analyses have shown that the variant virus (FS1001K) does not produce the 2.0-kb LAT or the 1.45- to 1.5-kb LAT either in vitro or in vivo; however, FS1001K produces an intact RL2 transcript in tissue culture. These data suggest that the 2.0-kb LAT putative ORF1 and ORF2 (or the first 1,186 bp of the 2.0-kb LAT) are dispensable for explant reactivation of latent HSV-1.

Characterization of an in vivo reactivation model of herpes simplex virus from mice trigeminal ganglia

Herpes simplex virus type 1 (HSV-1) is transcriptionally active during latent infection in human peripheral sensory ganglia. Viral gene expression includes the latency-associated transcripts (LATs) which have been linked to the ability of the virus to resume replication and reactivate. However, the molecular basis of reactivation and the mechanisms of action of these transcripts are unknown. In order to study these parameters, an in vivo reactivation model is needed. We investigated use of the mouse as the experimental animal, modifying the route of infection, the viral strain and the reactivation protocol. Following administration of human immunoglobulin 1 day prior to corneal infection, no infectious virus was detected in trigeminal ganglia (TG). However, latency was established in all infected animals as indicated by explant reactivation of TG, and in vivo reactivation was achieved in 30 to 40% of them. DNA quantification revealed that TG of immunized mice contained more HSV-1 DNA than did those of non-immunized mice. By in situ hybridization twice as many neuronal cells in TG of immunized mice were positive for LATs, compared with infected but non-immunized, mice. These findings suggest that suppression of primary infection facilitates reactivation by increasing HSV-1 copy number in latently infected nervous tissue.

Evidence for a novel regulatory pathway for herpes simplex virus gene expression in trigeminal ganglion neurons

Thymidine kinase (TK)-negative (TK-) mutant strains of herpes simplex virus type 1 (HSV-1) show reduced expression of alpha and beta viral genes during acute infection of trigeminal ganglion neurons following corneal infection (M. Kosz-Vnenchak, D. M. Coen, and D. M. Knipe, J. Virol. 64:5396-5402, 1990). It was surprising that a defect in a beta gene product would lead to decreased alpha and beta gene expression, given the regulatory pathways demonstrated for HSV infection of cultured cells. In this study, we have examined viral gene expression during reactivation from latent infection in explanted trigeminal ganglion tissue. In explant reactivation studies with wild-type virus, we observed viral productive gene expression over the first 48 h of explant incubation occurring in a temporal order (alpha, beta, gamma) similar to that in cultured cells. This occurred predominantly in latency-associated transcript-positive neurons but was limited to a fraction of these cells. In contrast, TK- mutant viruses showed greatly reduced alpha and beta gene expression upon explant of latently infected trigeminal ganglion tissue. An inhibitor of viral TK or an inhibitor of viral DNA polymerase greatly decreased viral lytic gene expression in trigeminal ganglion tissue latently infected with wild-type virus and explanted in culture. These results indicate that the regulatory mechanisms governing HSV gene expression are different in trigeminal ganglion neurons and cultured cells. We present a new model for viral gene expression in trigeminal ganglion neurons with implications for the nature of the decision process between latent infection and productive infection by HSV.

In vivo and in vitro reactivation impairment of a herpes simplex virus type 1 latency-associated transcript variant in a rabbit eye model.

Many recent studies of latent herpes simplex virus type 1 (HSV-1) infections within the nervous system have focused on the diploid genes encoding the latency-associated transcripts (LATs). The impaired explant reactivation of LAT variants from mouse trigeminal ganglia has implicated the LATs in the efficiency or speed of the reactivation process (D. A. Leib, C. L. Bogard, M. Kosz-Vnenchak, K. A. Hicks, D. M. Coen, D. M. Knipe, and P. A. Schaffer, J. Virol. 63:2893-2900, 1989; I. Steiner, J. G. Spivack, R. P. Lirette, S. M. Brown, A. R. MacLean, J. H. Subak-Sharpe, and N. W. Fraser, EMBO J. 8:505-511, 1989). However, it is not known how closely explant reactivation mimics the reactivation process in vivo. In the current study, a LAT variant (1704), parental strain (17+), and rescuant (1704R) were compared in vivo for reactivation of latent infection by iontophoresis in the rabbit eye model and in vitro by explant cocultivation of trigeminal ganglia from rabbits. Following iontophoresis, 17+ and 1704R reactivated in vivo from 76 and 64% of rabbits, respectively, while 1704 reactivated only from 4% (1 of 25) of the animals. In explant reactivation experiments, 17+ and 1704R reactivated from 98 and 67% of rabbit trigeminal ganglia, while 1704 reactivated from only 28% of trigeminal ganglia. The mean time required for the appearance of reactivated 1704 in explant culture, 17 days, was significantly longer than for 17+ and 1704R, 8 to 9 days. Thus, the explant reactivation kinetics in rabbit trigeminal ganglia reflect the behavior of LAT variant 1704 in vivo in the rabbit eye model. These data support the role of the LATs in the reactivation process and support the hypothesis that explant reactivation is a suitable system for analyzing the biological behavior of HSV-1 variants with defined genetic alterations in the LAT gene.

A herpes simplex virus type 1 latency-associated transcript mutant reactivates with normal kinetics from latent infection

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) accumulate in neuronal nuclei of latently infected ganglia. Explant reactivation kinetics of LAT deletion mutants in the mouse eye model have suggested a role for the LATs in the reactivation process. This report describes the construction and characterization of an HSV-1 strain HFEM mutant, TB1, disrupted within both copies of the LAT gene. TB1 contains a 440-base-pair segment of bacteriophage lambda DNA in place of a 168-base-pair deletion within the transcribed portion of the LAT gene. The 2.0-kilobase LAT was not produced after infection of tissue culture cells with TB1, but a 0.7- to 0.8-kilobase RNA was expressed. TB1 did establish latent infection after corneal inoculation as efficiently as the parental virus, and its reactivation kinetics from explanted ganglia were similar to those of HFEM. During latent infection with TB1, HSV-1 transcripts were not detectable. Rescuant virus (TB1-R) contained intact LAT genes, synthesized full-length LAT transcripts during productive infection in tissue culture, and reactivated from ganglionic explants of latently infected mice with normal kinetics. Thus, any function these transcripts have in the reactivation process appears to include the region between the putative LAT promoter and the disruption in TB1--a region of approximately 1,600 nucleotides, 800 of which encode the LATs.

Detection of HSV-1 proteins prior to the appearance of infectious virus in mouse trigeminal ganglia during reactivation of latent infection

Following corneal inoculation of mice HSV-1 produces an acute infection and establishes a latent infection in trigeminal ganglia. The latent virus can be reactivated in vitro by explantation of ganglionic tissue. Viral protein expression was studied in trigeminal ganglia during acute infection of mice and explant reactivation of latent infection. HSV-1 proteins were detectable by immunoprecipitation and immunostaining, in mouse ganglia only from 3–5 days post infection. Although during expiant reactivation it has been demonstrated that at 24 h post-explant the trigeminal ganglia are all infectious virus negative (Spivack, O'Boyle II and Fraser (1987) J. Virol. 61, 3288–3291), we have found that three HSV-1 proteins, of 175 kDa, 110 kDa and 90 kDa, are present in latently infected trigeminal ganglia as early as 6–21 h post explantation. Initially, only neuronal cells were positive by immunostaining with anti HSV-1 polyclonal serum for HSV-1 antigens, but at later times HSV-1 antigens were seen in non neuronal cells as well. These proteins may play a role in the initial stages of the reactivation process.

Herpes simplex virus type 1 latency-associated transcripts are evidently not essential for latent infection.

The herpes simplex virus type 1 (HSV-1) transcripts that can be detected during latent infection by Northern blot analysis in human and experimental animal sensory ganglia are encoded by diploid genes. To investigate their role in latent infection we studied HSV-1 variant 1704, which has deleted most of the IRL copy of the coding region of these RNAs and has a 1.2-kb deletion that is immediately upstream of the coding region of the TRL copy. During primary infection, 1704 replicated in trigeminal ganglia with kinetics similar to the parent virus (17+) and established latent infection. However, while explant reactivation of latent HSV-1 from trigeminal ganglia was detected in 100% of 17+ infected mice within 7 days, the reactivation of 1704 was significantly delayed, and 31 days elapsed before eight out of nine mice became virus positive. The recognized HSV-1 latency-associated RNAs were not detected during the latent state of 1704 by Northern blot analysis or in situ hybridization, which implies that the 1.2-kb deletion may contain the promoter or other important regulatory elements. The data indicate that detectable levels of these latency-associated transcripts are not required for viral replication, establishment, or maintenance (greater than 6 weeks) of HSV-1 latency in trigeminal ganglia, but suggest a role in reactivation.