Inclusion of the Guinea Pig Cytomegalovirus Pentameric Complex in a Live Virus Vaccine Aids Efficacy against Congenital Infection but Is Not Essential for Improving Maternal and Neonatal Outcomes.

The development of a vaccine against congenital human cytomegalovirus (HCMV) infection is a major priority. The pentameric complex (PC) of virion envelope proteins gH, gL, UL128, UL130, and UL131A is a key vaccine target. To determine the importance of immunity to the homologous PC encoded by guinea pig cytomegalovirus (GPCMV) in preventing congenital CMV, PC-intact and PC-deficient live-attenuated vaccines were generated and directly compared for immunogenicity and efficacy against vertical transmission in a vertical transmission model. A virulent PC-intact GPCMV (PC/intact) was modified by galK mutagenesis either to abrogate PC expression (PC/null; containing a frame-shift mutation in GP129, homolog of UL128) or to delete genes encoding three MHC Class I homologs and a protein kinase R (PKR) evasin while retaining the PC (3DX/Δ145). Attenuated vaccines were compared to sham immunization in a two-dose preconception subcutaneous inoculation regimen in GPCMV seronegative Hartley guinea pigs. Vaccines induced transient, low-grade viremia in 5/12 PC/intact-, 2/12 PC/null-, and 1/11 3DX/Δ145-vaccinated animals. Upon completion of the two-dose vaccine series, ELISA titers for the PC/intact group (geometic mean titer (GMT) 13,669) were not significantly different from PC/null (GMT 8127) but were significantly higher than for the 3DX/Δ145 group (GMT 6185; p < 0.01). Dams were challenged with salivary gland-adapted GPCMV in the second trimester. All vaccines conferred protection against maternal viremia. Newborn weights were significantly lower in sham-immunized controls (84.5 ± 2.4 g) compared to PC/intact (96 ± 2.3 g), PC/null (97.6 ± 1.9 g), or 3DX/Δ145 (93 ± 1.7) pups (p < 0.01). Pup mortality in sham-immunized controls was 29/40 (73%) and decreased to 1/44 (2.3%), 2/46 (4.3%), or 4/40 (10%) in PC/intact, PC/null, or 3DX/Δ145 groups, respectively (all p < 0.001 compared to control). Congenital GPCMV transmission occurred in 5/44 (11%), 16/46 (35%), or 29/38 (76%) of pups in PC/intact, PC/null, or 3DX/Δ145 groups, versus 36/40 (90%) in controls. For infected pups, viral loads were lower in pups born to vaccinated dams compared to controls. Sequence analysis demonstrated that infected pups in the vaccine groups had salivary gland-adapted GPCMV and not vaccine strain-specific sequences, indicating that congenital transmission was due to the challenge virus and not vaccine virus. We conclude that inclusion of the PC in a live, attenuated preconception vaccine improves immunogenicity and reduces vertical transmission, but PC-null vaccines are equal to PC-intact vaccines in reducing maternal viremia and protecting against GPCMV-related pup mortality.

A trimeric capable gB CMV vaccine provides limited protection against a highly cell associated and epithelial tropic strain of cytomegalovirus in guinea pigs.

Multiple strains of human cytomegalovirus (HCMV) can cause congenital cytomegalovirus (cCMV) by primary or secondary infection. The viral gB glycoprotein is a leading vaccine candidate, essential for infection of all cell-types, and immunodominant antibody target. Guinea pig cytomegalovirus (GPCMV) is the only small animal model for cCMV. Various gB vaccines have shown efficacy but studies have utilized truncated gB and protection against prototype strain 22122 with preferential tropism to fibroblasts despite encoding a gH-based pentamer complex for non-fibroblast infection. A highly cell-associated novel strain of GPCMV (TAMYC) with 99 % identity in gB sequence to 22122 exhibited preferred tropism to epithelial cells. An adenovirus vaccine encoding full-length gB (AdgB) was highly immunogenic and partially protected against 22122 strain challenge in vaccinated animals but not when challenged with TAMYC strain. GPCMV studies with AdgB vaccine sera on numerous cell-types demonstrated impaired neutralization (NA50) compared to fibroblasts. GPCMV-convalescent sera including pentamer complex antibodies increased virus neutralization on non-fibroblasts and anti-gB depletion from GPCMV-convalescent sera had minimal impact on epithelial cell neutralization. GPCMV(PC+) 22122-convalescent animals challenged with TAMYC exhibited higher protection compared to AdgB vaccine. Overall, results suggest that antibody response to both gB and PC are important components of a GPCMV vaccine.

Identification and functional analyses of a cell-death inhibitor encoded by guinea pig cytomegalovirus gp38.1 in cell culture and in animals.

Cytomegaloviruses (CMVs) employ an array of strategies designed to interfere with host defence responses against pathogens. Studies on such evasion mechanisms are important for understanding the pathogenesis of CMV diseases. Although guinea pig CMV (GPCMV) provides a useful animal model for congenital CMV infection, its evasion strategies are not fully elucidated. Here, we analysed a genome locus that may encode gene products for the GPCMV evasion mechanisms and found the following. (1) RACE analyses identified five transcripts in the GP38-gp38.4 locus, one of which was a spliced product encoding gp38.1. Similarities in the splicing pattern and gene position of gp38.1 to human CMV UL37 and its exon 1 encoding vMIA (viral mitochondria-localized inhibitor of apoptosis) suggest that the gp38.1 gene encodes an apoptosis inhibitor. (2) In a transient transfection assay, gp38.1 localized in the mitochondria and relocated BAX from the cytoplasm to the mitochondria, although its co-localization with BAK was not evident. Further, the expression of gp38.1 partially reduced staurosporine-induced apoptosis. (3) GPCMV defective in the gp38.1 ORF (Δ38.1) and the virus that rescues the defect (r38.1) were generated. Guinea pig fibroblast cells infected with Δ38.1 died earlier than r38.1-infected cells, which resulted in the lower yields of Δ38.1. (4) In animals, viral loads in the spleens of r38.1-infected guinea pigs were higher than those in the spleens of Δ38.1-infected animals. In conclusion, although GPCMV gp38.1 exerts a vMIA-like function, its inhibitory effect was not robust, suggesting the presence of additional inhibitory molecule(s), such as a BAK-specific inhibitor.

Comparative Analysis of Roseoloviruses in Humans, Pigs, Mice, and Other Species.

Viruses of the genus Roseolovirus belong to the subfamily Betaherpesvirinae, family Herpesviridae. Roseoloviruses have been studied in humans, mice and pigs, but they are likely also present in other species. This is the first comparative analysis of roseoloviruses in humans and animals. The human roseoloviruses human herpesvirus 6A (HHV-6A), 6B (HHV-6B), and 7 (HHV-7) are relatively well characterized. In contrast, little is known about the murine roseolovirus (MRV), also known as murine thymic virus (MTV) or murine thymic lymphotrophic virus (MTLV), and the porcine roseolovirus (PRV), initially incorrectly named porcine cytomegalovirus (PCMV). Human roseoloviruses have gained attention because they can cause severe diseases including encephalitis in immunocompromised transplant and AIDS patients and febrile seizures in infants. They have been linked to a number of neurological diseases in the immunocompetent including multiple sclerosis (MS) and Alzheimer's. However, to prove the causality in the latter disease associations is challenging due to the high prevalence of these viruses in the human population. PCMV/PRV has attracted attention because it may be transmitted and pose a risk in xenotransplantation, e.g., the transplantation of pig organs into humans. Most importantly, all roseoloviruses are immunosuppressive, the humoral and cellular immune responses against these viruses are not well studied and vaccines as well as effective antivirals are not available.

Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig.

Guinea pig cytomegalovirus (GPCMV) encodes a homolog pentameric complex (PC) for specific cell tropism and congenital infection. In human cytomegalovirus, the PC is an important antibody neutralizing target and GPCMV studies will aid in the development of intervention strategies. Deletion mutants of the C-terminal domains of unique PC proteins (UL128, UL130 and UL131 homologs) were unable to form a PC in separate transient expression assays. Minor modifications to the UL128 homolog (GP129) C-terminal domain enabled PC formation but viruses encoding these mutants had altered tropism to renal and placental trophoblast cells. Mutation of the presumptive CC chemokine motif encoded by GP129 was investigated by alanine substitution of the CC motif (codons 26-27) and cysteines (codons 47 and 62). GP129 chemokine mutants formed PC but GP129 chemokine mutant viruses had reduced epitropism. A GP129 chemokine mutant virus pathogenicity study demonstrated reduced viral load to target organs but highly extended viremia.

A Novel Non-Replication-Competent Cytomegalovirus Capsid Mutant Vaccine Strategy Is Effective in Reducing Congenital Infection.

UNLABELLED: Congenital cytomegalovirus (CMV) infection is a leading cause of mental retardation and deafness in newborns. The guinea pig is the only small animal model for congenital CMV infection. A novel CMV vaccine was investigated as an intervention strategy against congenital guinea pig cytomegalovirus (GPCMV) infection. In this disabled infectious single-cycle (DISC) vaccine strategy, a GPCMV mutant virus was used that lacked the ability to express an essential capsid gene (the UL85 homolog GP85) except when grown on a complementing cell line. In vaccinated animals, the GP85 mutant virus (GP85 DISC) induced an antibody response to important glycoprotein complexes considered neutralizing target antigens (gB, gH/gL/gO, and gM/gN). The vaccine also generated a T cell response to the pp65 homolog (GP83), determined via a newly established guinea pig gamma interferon enzyme-linked immunosorbent spot assay. In a congenital infection protection study, GP85 DISC-vaccinated animals and a nonvaccinated control group were challenged during pregnancy with wild-type GPCMV (10(5) PFU). The pregnant animals carried the pups to term, and viral loads in target organs of pups were analyzed. Based on live pup births in the vaccinated and control groups (94.1% versus 63.6%), the vaccine was successful in reducing mortality (P = 0.0002). Additionally, pups from the vaccinated group had reduced CMV transmission, with 23.5% infected target organs versus 75.9% in the control group. Overall, these preliminary studies indicate that a DISC CMV vaccine strategy has the ability to induce an immune response similar to that of natural virus infection but has the increased safety of a non-replication-competent virus, which makes this approach attractive as a CMV vaccine strategy. IMPORTANCE: Congenital CMV infection is a leading cause of mental retardation and deafness in newborns. An effective vaccine against CMV remains an elusive goal despite over 50 years of CMV research. The guinea pig, with a placenta structure similar to that in humans, is the only small animal model for congenital CMV infection and recapitulates disease symptoms (e.g., deafness) in newborn pups. In this report, a novel vaccine strategy against congenital guinea pig cytomegalovirus (GPCMV) infection was developed, characterized, and tested for efficacy. This disabled infectious single-cycle (DISC) vaccine strategy induced a neutralizing antibody or a T cell response to important target antigens. In a congenital infection protection study, animals were protected against CMV in comparison to the nonvaccinated group (52% reduction of transmission). This novel vaccine was more effective than previously tested gB-based vaccines and most other strategies involving live virus vaccines. Overall, the DISC vaccine is a safe and promising approach against congenital CMV infection.

Roseoloviruses manipulate host cell cycle.

During lytic infections HHV-6A and HHV-6B disrupt E2F1-Rb complexes by Rb degradation, releasing E2F1 and driving the infected cells toward the S-phase. Whereas upon infection E2F1 and its cofactor DP1 were up-regulated, additional E2F responsive genes were expressed differentially in various cells. E2F binding sites were identified in promoters of several HHV-6 genes, including the U27 and U79 associated with viral DNA replication, revealing high dependence on the binding site and the effect of the E2F1 transcription factor. Viral genes regulation by E2F1 can synchronize viral replication with the optimal cell cycle phase, enabling utilization of host resources for successful viral replication. Furthermore, it was found that infection by roseoloviruses leads to cell cycle arrest, mostly in the G2/M-phase.

Roseoloviruses: unmet needs and research priorities: perspective.

The human roseoloviruses, human herpesviruses 6A (HHV-6A), HHV-6B, and HHV-7, are highly prevalent viruses that typically cause fever/rash illnesses such as roseola during early life primary infections. They also cause significant neurologic disease and complications following stem cell and solid organ transplantation, and have suggestive but less certain etiologic associations with other neurologic diseases and immunologic disorders. The US National Institute of Allergy and Infectious Diseases recently sponsored a workshop (Roseoloviruses: Clinical Impact, Interventions, and Research Needs) to discuss disease associations, novel biology, and the many unmet research needs related to Roseoloviruses. This perspective is a distillation of the workshop's presentations and discussions, with a focus on the more general research priorities that emerged.

Roseomics: a blank slate.

Recent technological advances have led to an explosion in the system-wide profiling of biological processes in the study of herpesvirus biology, herein referred to as '-omics'. In many cases these approaches have revealed novel virus-induced changes to host cell biology that can be targeted with new antiviral therapeutics. Despite these successes, -omics approaches are not widely applied in the study of roseoloviruses. Here we describe examples of how -omics studies have shaped our understanding of herpesvirus biology, and discuss how these approaches might be used to identify host and viral factors that mediate roseolovirus pathogenesis.

The search for circulating epilepsy biomarkers.

Few would experience greater benefit from the development of biomarkers than those who suffer from epilepsy. Both the timing of individual seizures and the overall course of the disease are highly unpredictable, and the associated morbidity is considerable. Thus, there is an urgent need to develop biomarkers that can predict the progression of epilepsy and treatment response. Doing so may also shed light on the mechanisms of epileptogenesis and pharmacoresistance, which remain elusive despite decades of study. However, recent advances suggest the possible identification of circulating epilepsy biomarkers - accessible in blood, cerebrospinal fluid or urine. In this review, we focus on advances in several areas: neuroimmunology and inflammation; neurological viral infection; exemplary pediatric syndromes; and the genetics of pharmacoresistance, as relevant to epilepsy. These are fertile areas of study with great potential to yield accessible epilepsy biomarkers.

Guinea pig cytomegalovirus GP129/131/133, homologues of human cytomegalovirus UL128/130/131A, are necessary for infection of monocytes and macrophages.

The GP129, GP131 and GP133 genes of guinea pig cytomegalovirus (GPCMV) are homologues of human cytomegalovirus UL128, UL130 and UL131A, respectively, which are essential for infection of endothelial and epithelial cells, and for viral transmission to leukocytes. Our previous study demonstrated that a GPCMV strain lacking the 1.6 kb locus that contains the GP129, GP131 and GP133 genes had a growth defect in animals. Here, we demonstrated that the WT strain, but not the 1.6 kb-deleted strain, formed capsids in macrophages prepared from the peritoneal fluid. To understand the mechanism, we prepared GPCMV strains defective in each of GP129, GP131 and GP133, and found that they were all essential for the infection of peritoneal, splenic and PBMC-derived macrophages/monocytes, and for expression of immediate-early antigens in the macrophages/monocytes, although they were dispensable for infection of fibroblasts. Monocyte/macrophage tropism could be one of the important determinants for viral dissemination in vivo.

Molecular and biological characterization of a new isolate of guinea pig cytomegalovirus.

Development of a vaccine against congenital infection with human cytomegalovirus is complicated by the issue of re-infection, with subsequent vertical transmission, in women with pre-conception immunity to the virus. The study of experimental therapeutic prevention of re-infection would ideally be undertaken in a small animal model, such as the guinea pig cytomegalovirus (GPCMV) model, prior to human clinical trials. However, the ability to model re-infection in the GPCMV model has been limited by availability of only one strain of virus, the 22122 strain, isolated in 1957. In this report, we describe the isolation of a new GPCMV strain, the CIDMTR strain. This strain demonstrated morphological characteristics of a typical Herpesvirinae by electron microscopy. Illumina and PacBio sequencing demonstrated a genome of 232,778 nt. Novel open reading frames ORFs not found in reference strain 22122 included an additional MHC Class I homolog near the right genome terminus. The CIDMTR strain was capable of dissemination in immune compromised guinea pigs, and was found to be capable of congenital transmission in GPCMV-immune dams previously infected with salivary gland­adapted strain 22122 virus. The availability of a new GPCMV strain should facilitate study of re-infection in this small animal model.

Characterization of the guinea pig CMV gH/gL/GP129/GP131/GP133 complex in infection and spread.

In human cytomegalovirus (HCMV), the UL128-131A locus plays an essential role in cellular tropism and spread. Here, we report the complete annotation of the GP129-133 locus from guinea pig cytomegalovirus (GPCMV) and the discovery of the UL131A homolog, named GP133. We have found that similar to HCMV the GP129-133 proteins form a pentamer complex with the GPCMV glycoproteins gH and gL. In addition, we find that the GP129-133 proteins play a critical role in entry as the GP129-133 deletion mutant shows a defect in both endothelial and fibroblast cell entry. Although the GP129-133 deletion strain can propagate in vitro, we find that the deletion fails to spread in vivo. Interestingly, the wildtype strain can spontaneously give rise to the GP129-133 deletion strain during in vivo spread, suggesting genetic instability at this locus.

The impact of genome length on replication and genome stability of the herpesvirus guinea pig cytomegalovirus.

The impact of genome length on replication and genome stability was assessed for guinea pig cytomegalovirus (GPCMV), a member of the Herpesviridae. The 233-kb genome could be decreased by 15.1 kb without discernable impact on viral replication efficiency in vitro. Viruses with genomes under-length by up to 31 kb replicated with decreased efficiencies but this appeared to arise from the loss of augmenting viral genes rather than decreased genome length. Two deletions that were non-lethal on their own were lethal when combined, suggesting that the resulting 40.1 kb under-length genome fell below a minimum packageable size. Genomes over-length by 8.8 kb gave rise to spontaneous deletions just to the right of the major immediate early locus, the same region that undergoes deletions during fibroblast passage of human and rhesus cytomegaloviruses. These results suggest that genome integrity should be confirmed for herpesvirus mutants in which genome length is increased even modestly.

Establishment of a cell-based assay for screening of compounds inhibiting very early events in the cytomegalovirus replication cycle and characterization of a compound identified using the assay.

To simplify the detection of infectious human cytomegalovirus (HCMV), we generated a cell line that produced luciferase in a dose-dependent manner upon HCMV infection. Using this cell line, we identified anti-HCMV compounds from a diverse library of 9,600 compounds. One of them, 1-(3,5-dichloro-4-pyridyl)piperidine-4-carboxamide (DPPC), was effective against HCMV (Towne strain) infection of human lung fibroblast cells at a 50% effective concentration of 2.5 microM. DPPC also inhibited the growth of clinical HCMV isolates and guinea pig and mouse cytomegaloviruses. Experiments using various time frames for treatment of the cells with DPPC demonstrated that DPPC was effective during the first 24 h after HCMV infection. DPPC treatment decreased not only viral DNA replication but also IE1 and IE2 expression at mRNA and protein levels in the HCMV-infected cells. However, DPPC did not inhibit the attachment of HCMV particles to the cell surface. DPPC is a unique compound that targets the very early phase of cytomegalovirus infection, probably by disrupting a pathway that is important after viral entry but before immediate-early gene expression.

Pathogenesis of cytomegalovirus-associated labyrinthitis in a guinea pig model.

Cytomegalovirus infects fetuses through the placenta, resulting in various congenital disorders in newborns, including hearing loss. We developed a monoclonal antibody to guinea pig cytomegalovirus (GPCMV) that was available for immunohistochemistry, and investigated the expression of the GPCMV antigen in animal models of direct and congenital infections. Injection of GPCMV, directly to the inner ear, increased the sound pressure level and resulted in labyrinthitis with severe inflammation. Immunohistochemistry detected GPCMV-infected cells mainly in the scala tympani, scala vestibule and spinal ganglion, but rarely in the cochlear duct. Injection of GPCMV to 5-week pregnant guinea pigs resulted in severe labyrinthitis in fetuses. Immunohistochemistry detected GPCMV-infected cells in the perilymph area and spinal ganglion, but not in the endolymph area, including hair cells. These data suggest that the virus spreads via the perilymph and neural routes in the inner ear of both models of direct and congenital infections.

Molecular, biological, and in vivo characterization of the guinea pig cytomegalovirus (CMV) homologs of the human CMV matrix proteins pp71 (UL82) and pp65 (UL83).

We recently identified the genes encoding the guinea pig cytomegalovirus (GPCMV) homologs of the upper and lower matrix proteins of human CMV, pp71 (UL82) and pp65 (UL83), which we designated GP82 and GP83, respectively. Transient-expression studies with a GP82 plasmid demonstrated that the encoded protein targets the nucleus and that the infectivity and plaquing efficiency of cotransfected GPCMV viral DNA was enhanced by GP82. The transactivation function of GP82 was not limited to GPCMV, but was also observed for a heterologous virus, herpes simplex virus type 1 (HSV-1). This was confirmed by its ability to complement the growth of an HSV-1 VP16 transactivation-defective mutant virus in an HSV viral DNA cotransfection assay. Study of a GP82 "knockout" virus (and its attendant rescuant), generated on a GPCMV bacterial artificial chromosome construct, confirmed the essential nature of the gene. Conventional homologous recombination was used to generate a GP83 mutant to examine the role of GP83 in the viral life cycle. Comparison of the one-step growth kinetics of the GP83 mutant (vAM409) and wild-type GPCMV indicated that GP83 protein is not required for viral replication in tissue culture. The role of GP83 in vivo was examined by comparing the pathogenesis of wild-type GPCMV, vAM409, and a control virus, vAM403, in guinea pigs. The vAM409 mutant was significantly attenuated for dissemination in immunocompromised strain 2 guinea pigs, suggesting that the GP83 protein is essential for full pathogenicity in vivo.

Dramatic effects of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole riboside on the genome structure, packaging, and egress of guinea pig cytomegalovirus.

The halogenated benzimidazoles BDCRB (2-bromo-5,6-dichloro-1-beta-D-riborfuranosyl benzimidazole riboside) and TCRB (2,5,6-trichloro-1-beta-D-riborfuranosyl benzimidazole riboside) were the first compounds shown to inhibit cleavage and packaging of herpesvirus genomes. Both inhibit the formation of unit length human cytomegalovirus (HCMV) genomes by a poorly understood mechanism (M. R. Underwood et al., J. Virol. 72:717-715, 1998; P. M. Krosky et al., J. Virol. 72:4721-4728, 1998). Because the simple genome structure of guinea pig cytomegalovirus (GPCMV) provides a useful model for the study of herpesvirus DNA packaging, we investigated the effects of BDCRB on GPCMV. GPCMV proved to be sensitive to BDCRB (50% inhibitory concentration = 4.7 microM), although somewhat less so than HCMV. In striking contrast to HCMV, however, a dose of BDCRB sufficient to reduce GPCMV titers by 3 logs (50 microM) had no effect on the quantity of GPCMV genomic DNA that was formed in infected cells. Electron microscopy revealed that this DNA was in fact packaged within intranuclear capsids, but these capsids failed to egress from the nucleus and failed to protect the DNA from nuclease digestion. The terminal structure of genomes formed in the presence of BDCRB was also altered. Genomes with ends lacking a terminal repeat at the right end, which normally exist in an equimolar ratio with those having one copy of the repeat at the right end, were selectively eliminated by BDCRB treatment. At the left end, BDCRB treatment appeared to induce heterogeneous truncations such that 2.7 to 4.9 kb of left-end-terminal sequences were missing. These findings suggest that BDCRB induces premature cleavage events that result in truncated genomes packaged within capsids that are permeable to nuclease. Based on these and other observations, we propose a model for duplication of herpesvirus terminal repeats during the cleavage and packaging process that is similar to one proposed for bacteriophage T7 (Y. B. Chung, C. Nardone, and D. C. Hinkle, J. Mol. Biol. 216:939-948, 1990).