Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation.

BACKGROUND: Porcine cytomegalovirus (PCMV) is a porcine roseolovirus (PCMV/PRV) which is widely distributed in pigs. Transmission of PCMV/PRV in preclinical xenotransplantations was shown to significantly reduce the survival time of the pig transplants in non-human primates. PCMV/PRV was also transmitted in the first transplantation of a pig heart into a human patient. To analyze how PCMV/PRV could be introduced into pig breeds, especially considering cloned transgenic pigs, and subsequently spread in breeding facilities, we screened ovaries and derived materials which are used to perform somatic cell nuclear transfer (SCNT). METHODS: DNA was isolated from ovarian tissues, follicular fluids, oocytes with cumulus cells, denuded oocytes and parthenotes. A real-time PCR with PCMV/PRV-specific primers and a probe was performed to detect PCMV/PRV. Furthermore, a Western blot assay using a recombinant fragment of the gB protein of PCMV/PRV was performed to screen for virus-specific antibodies in the follicular fluids. RESULTS: PCMV/PRV was found by real-time PCR in ovarian tissues, in the follicular fluid and in oocytes. In parthenotes the virus could not be detected, most-likely due to the low amount of DNA used. By Western blot assay specific antibodies against PCMV/PRV were found in 19 of 20 analyzed follicular fluids. CONCLUSION: PCMV/PRV was found in ovarian tissues, in the follicular fluids and also in denuded oocytes, indicating that the virus is present in the animals of which the oocytes were taken from. Despite several washing steps of the denuded oocytes, which are subsequently used for microinjection or SCNT, the virus could still be detected. Therefore, the virus could infect oocytes during genetic modifications or stay attached to the surface of the oocytes, potentially infecting SCNT recipient animals.

A Fully Protective Congenital CMV Vaccine Requires Neutralizing Antibodies to Viral Pentamer and gB Glycoprotein Complexes but a pp65 T-Cell Response Is Not Necessary.

A vaccine against congenital cytomegalovirus infection is a high priority. Guinea pig cytomegalovirus (GPCMV) is the only congenital CMV small animal model. GPCMV encodes essential glycoprotein complexes for virus entry (gB, gH/gL/gO, gM/gN) including a pentamer complex (gH/gL/GP129/GP131/GP133 or PC) for endocytic cell entry. The cohorts for protection against congenital CMV are poorly defined. Neutralizing antibodies to the viral glycoprotein complexes are potentially more important than an immunodominant T-cell response to the pp65 protein. In GPCMV, GP83 (pp65 homolog) is an evasion factor, and the GP83 mutant GPCMV has increased sensitivity to type I interferon. Although GP83 induces a cell-mediated response, a GP83-only-based vaccine strategy has limited efficacy. GPCMV attenuation via GP83 null deletion mutant in glycoprotein PC positive or negative virus was evaluated as live-attenuated vaccine strains (GP83dPC+/PC-). Vaccinated animals induced antibodies to viral glycoprotein complexes, and PC+ vaccinated animals had sterilizing immunity against wtGPCMV challenge. In a pre-conception vaccine (GP83dPC+) study, dams challenged mid-2nd trimester with wtGPCMV had complete protection against congenital CMV infection without detectable virus in pups. An unvaccinated control group had 80% pup transmission rate. Overall, gB and PC antibodies are key for protection against congenital CMV infection, but a response to pp65 is not strictly necessary.

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.

Differences in the effects of mutations in GP131, a guinea pig cytomegalovirus homologue of pentameric complex component UL130, on macrophage and epithelial cell infection.

As congenital cytomegalovirus (CMV) infection is the major cause of developmental abnormalities in children, the development of effective vaccines is critical to public health. Recent studies have demonstrated that the pentameric complex (Pentamer) of glycoproteins, which is required for human CMV infection of endothelial and epithelial cells, could be a potent vaccine antigen. As guinea pig CMV (GPCMV) infects congenitally and encodes homologues of all Pentamer components, GPCMV models are considered to be useful for the development of vaccine strategies. Here, to clarify the precise requirement of GP131, one of the GPCMV Pentamer components, for the infection of epithelial cells and macrophages, we prepared several mutants with a charged amino acid-to-alanine alteration in GP131 and found some differences in the effects of the mutations on the infection of the two cell types, suggesting the existence of cell type-dependent recognition or function of Pentamer in GPCMV infection.

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.

The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting.

Guinea pig cytomegalovirus (GPCMV) immediate early proteins, IE1 and IE2, demonstrated structural and functional homologies with human cytomegalovirus (HCMV). GPCMV IE1 and IE2 co-localized in the nucleus with each other, the viral polymerase and guinea pig ND10 components (gpPML, gpDaxx, gpSp100, gpATRX). IE1 showed direct interaction with ND10 components by immunoprecipitation unlike IE2. Additionally, IE1 protein disrupted ND10 bodies. IE1 mutagenesis mapped the nuclear localization signal to the C-terminus and identified the core domain for gpPML interaction. Individual knockout of GPCMV GP122 or GP123 (IE2 and IE1 unique exons respectively) was lethal to the virus. However, an IE1 mutant (codons 234-474 deleted), was viable with attenuated viral growth kinetics and increased susceptibility to type I interferon (IFN-I). In HCMV, the IE proteins are important T cell target antigens. Consequently, characterization of the homologs in GPCMV provides a basis for their evaluation in candidate vaccines against congenital infection.

Identification of Novel Betaherpesviruses in Iberian Bats Reveals Parallel Evolution.

A thorough search for bat herpesviruses was carried out in oropharyngeal samples taken from most of the bat species present in the Iberian Peninsula from the Vespertilionidae, Miniopteridae, Molossidae and Rhinolophidae families, in addition to a colony of captive fruit bats from the Pteropodidae family. By using two degenerate consensus PCR methods targeting two conserved genes, distinct and previously unrecognized bat-hosted herpesviruses were identified for the most of the tested species. All together a total of 42 potentially novel bat herpesviruses were partially characterized. Thirty-two of them were tentatively assigned to the Betaherpesvirinae subfamily while the remaining 10 were allocated into the Gammaherpesvirinae subfamily. Significant diversity was observed among the novel sequences when compared with type herpesvirus species of the ICTV-approved genera. The inferred phylogenetic relationships showed that most of the betaherpesviruses sequences fell into a well-supported unique monophyletic clade and support the recognition of a new betaherpesvirus genus. This clade is subdivided into three major clades, corresponding to the families of bats studied. This supports the hypothesis of a species-specific parallel evolution process between the potentially new betaherpesviruses and their bat hosts. Interestingly, two of the betaherpesviruses' sequences detected in rhinolophid bats clustered together apart from the rest, closely related to viruses that belong to the Roseolovirus genus. This suggests a putative third roseolo lineage. On the contrary, no phylogenetic structure was detected among several potentially novel bat-hosted gammaherpesviruses found in the study. Remarkably, all of the possible novel bat herpesviruses described in this study are linked to a unique bat species.

Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired during Fibroblast Passage Restores Pathogenesis in Immune-Suppressed Guinea Pigs and in the Context of Congenital Infection.

UNLABELLED: Guinea pig cytomegalovirus (GPCMV) provides a valuable model for congenital cytomegalovirus transmission. Salivary gland (SG)-passaged stocks of GPCMV are pathogenic, while tissue culture (TC) passage in fibroblasts results in attenuation. Nonpathogenic TC-derived virus N13R10 (cloned as a bacterial artificial chromosome [BAC]) has a 4-bp deletion that disrupts GP129, which encodes a subunit of the GPCMV pentameric complex (PC) believed to govern viral entry into select cell types, and GP130, an overlapping open reading frame (ORF) of unknown function. To determine if this deletion contributes to attenuation of N13R10, markerless gene transfer in Escherichia coli was used to construct virus r129, a variant of N13R10 in which the 4-bp deletion is repaired. Virions from r129 were found to contain GP129 as well as two other PC subunit proteins, GP131 and GP133, whereas these three PC subunits were absent from N13R10 virions. Replication of r129 in fibroblasts appeared unaltered compared to that of N13R10. However, following experimental challenge of immunocompromised guinea pigs, r129 induced significant weight loss, longer duration of viremia, and dramatically higher (up to 1.5 × 10(6)-fold) viral loads in blood and end organs compared to N13R10. In pregnant guinea pigs, challenge with doses of r129 virus of ≥5 × 10(6) PFU resulted in levels of maternal viremia, congenital transmission, pup viral loads, intrauterine growth restriction, and pup mortality comparable to that induced by pathogenic SG virus, although higher doses of r129 were required. These results suggest that the GP129-GP130 mutation is a significant contributor to attenuation of N13R10, likely by abrogating expression of a functional PC. IMPORTANCE: Tissue culture adaptation of cytomegaloviruses rapidly selects for mutations, deletions, and rearrangements in the genome, particularly for viruses passaged in fibroblast cells. Some of these mutations are focused in the region of the genome encoding components of the pentameric complex (PC), in particular homologs of human cytomegalovirus (HCMV) proteins UL128, UL130, and UL131A. These mutations can attenuate the course of infection when the virus is reintroduced into animals for vaccine and pathogenesis studies. This study demonstrates that a deletion that arose during the process of tissue culture passage can be repaired, with subsequent restoration of pathogenicity, using BAC-based mutagenesis. Restoration of pathogenicity by repair of a frameshift mutation in GPCMV gene GP129 using this approach provides a valuable genetic platform for future studies using the guinea pig model of congenital CMV infection.

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.

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.

Past, present, and future perspectives on the diagnosis of Roseolovirus infections.

Diagnosis of Roseolovirus infections mandates careful selection of patients, samples, and testing methods. We review advances in the field and highlight research priorities. Quantitative (q)PCR can accurately identify and distinguish between human herpesvirus 6 (HHV-6) species A and B. Whether screening of high-risk patients improves outcomes is unclear. Chromosomally integrated (ci)HHV-6 confounds test interpretation but can be ruled out with digital PCR. Reverse transcription qPCR may be a more specific and clinically applicable test for actively replicating Roseoloviruses, particularly among patients with ciHHV-6. Interpretation of Roseolovirus test results faces many challenges. However, careful application of refined and emerging diagnostic techniques will allow for increasingly accurate diagnosis of clinically significant infections and disease associations.

African great apes are naturally infected with roseoloviruses closely related to human herpesvirus 7.

UNLABELLED: Primates are naturally infected with herpesviruses. During the last 15 years, the search for homologues of human herpesviruses in nonhuman primates allowed the identification of numerous viruses belonging to the different herpesvirus subfamilies and genera. No simian homologue of human herpesvirus 7 (HHV7) has been reported to date. To investigate the putative existence of HHV7-like viruses in African great apes, we applied the consensus-degenerate hybrid oligonucleotide primers (CODEHOP) program-mediated PCR strategy to blood DNA samples from the four common chimpanzee subspecies (Pan troglodytes verus, P. t. ellioti, P. t. troglodytes, and P. t. schweinfurthii), pygmy chimpanzees (Pan paniscus), as well as lowland gorillas (Gorilla gorilla gorilla). This study led to the discovery of a novel roseolovirus close to HHV7 in each of these nonhuman primate species and subspecies. Generation of the partial glycoprotein B (1,111-bp) and full-length DNA polymerase (3,036/3,042-bp) gene sequences allowed the deciphering of their evolutionary relationships. Phylogenetic analyses revealed that HHV7 and its African great ape homologues formed well-supported monophyletic lineages whose topological resemblance to the host phylogeny is suggestive of virus-host codivergence. Notably, the evolutionary branching points that separate HHV7 from African great ape herpesvirus 7 are remarkably congruent with the dates of divergence of their hosts. Our study shows that African great apes are hosts of human herpesvirus homologues, including HHV7 homologues, and that the latter, like other DNA viruses that establish persistent infections, have cospeciated with their hosts. IMPORTANCE: Human herpesviruses are known to possess simian homologues. However, surprisingly, none has been identified to date for human herpesvirus 7 (HHV7). This study is the first to describe simian homologues of HHV7. The extensive search performed on almost all African great ape species and subspecies, i.e., common chimpanzees of the four subspecies, bonobos, and lowland gorillas, has allowed characterization of a specific virus in each. Genetic characterization of the partial glycoprotein B and full-length DNA polymerase gene sequences, followed by their phylogenetic analysis and estimation of divergence times, has shed light on the evolutionary relationships of these viruses. In this respect, we conclusively demonstrate the cospeciation between these new viruses and their hosts and report cases of cross-species transmission between two common chimpanzee subspecies in both directions.

The first endogenous herpesvirus, identified in the tarsier genome, and novel sequences from primate rhadinoviruses and lymphocryptoviruses.

Herpesviridae is a diverse family of large and complex pathogens whose genomes are extremely difficult to sequence. This is particularly true for clinical samples, and if the virus, host, or both genomes are being sequenced for the first time. Although herpesviruses are known to occasionally integrate in host genomes, and can also be inherited in a Mendelian fashion, they are notably absent from the genomic fossil record comprised of endogenous viral elements (EVEs). Here, we combine paleovirological and metagenomic approaches to both explore the constituent viral diversity of mammalian genomes and search for endogenous herpesviruses. We describe the first endogenous herpesvirus from the genome of the Philippine tarsier, belonging to the Roseolovirus genus, and characterize its highly defective genome that is integrated and flanked by unambiguous host DNA. From a draft assembly of the aye-aye genome, we use bioinformatic tools to reveal over 100,000 bp of a novel rhadinovirus that is the first lemur gammaherpesvirus, closely related to Kaposi's sarcoma-associated virus. We also identify 58 genes of Pan paniscus lymphocryptovirus 1, the bonobo equivalent of human Epstein-Barr virus. For each of the viruses, we postulate gene function via comparative analysis to known viral relatives. Most notably, the evidence from gene content and phylogenetics suggests that the aye-aye sequences represent the most basal known rhadinovirus, and indicates that tumorigenic herpesviruses have been infecting primates since their emergence in the late Cretaceous. Overall, these data show that a genomic fossil record of herpesviruses exists despite their extremely large genomes, and expands the known diversity of Herpesviridae, which will aid the characterization of pathogenesis. Our analytical approach illustrates the benefit of intersecting evolutionary approaches with metagenomics, genetics and paleovirology.

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.

Detection of five rash-associated viruses using multiplex real-time PCR during 2006-2011.

Many viruses have been reported to be associated with rash development. Multiplex real-time PCR was used to investigate the presence of 5 viruses associated with rashes: measles virus (MV), rubella virus (RV), human parvovirus B19 (PVB19), human herpes virus 6 (HHV-6), and HHV-7. A total of 187 clinical specimens from 169 patients with erythema were collected between January 2006 and December 2011. Virus-positive specimens were as follows: MV (n = 23), PVB19 (n = 8), RV (n = 2), HHV-6 (n = 5), HHV-7 (n = 1), MV and PVB19 (n = 1), and HHV-6 and HHV-7 (n = 1). All of the MV-positive specimens were collected in 2007 and the strains whose sequence were available (21/24, 87.5%) were of genotype D5. The results indicate that multiplex real-time PCR might be a useful screening method for detecting and differentiating rash-associated viruses in clinical specimens.

Antagonism of the protein kinase R pathway by the guinea pig cytomegalovirus US22-family gene gp145.

Viral double-stranded RNA (dsRNA) activates protein kinase R (PKR), which phosphorylates eIF2α and inhibits translation. In response, viruses have evolved various strategies to evade the antiviral impact of PKR. We investigated whether guinea pig cytomegalovirus (GPCMV), a useful model of congenital CMV infection, encodes a gene that interferes with the PKR pathway. Using a proteomic screen, we identified several GPCMV dsRNA-binding proteins, among which only gp145 rescued replication of a vaccinia virus mutant that lacks E3L. gp145 also reversed the inhibitory effects of PKR on expression of a cotransfected reporter gene. Mapping studies demonstrated that the gp145 dsRNA-binding domain has homology to the PKR antagonists of other CMVs. However, dsRNA-binding by gp145 is not sufficient for it to block PKR. gp145 differs from the PKR antagonists of murine CMV in that it functions alone and from those encoded by human CMV in functioning in cells from both primates and rodents.

Identification of human herpesviruses 1 to 8 in Tunisian multiple sclerosis patients and healthy blood donors.

Members of the human Herpesviridae family are candidates for representing the macroenvironmental factors associated with multiple sclerosis (MS) pathogenesis. To verify the possible role of human herpesviruses (HHVs) as triggering or aggravating factors in relapsing-remitting multiple sclerosis clinical outcome, we studied the prevalence of all eight human herpesviruses in whole blood samples collected from 51 MS patients and from 51 healthy controls. The presence of DNA of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7) and human herpesvirus 8 (HHV-8) was searched by specific nested polymerase chain reaction. HHVs were significantly more prevalent in the blood of MS patients than in those of the controls (P < 10(-4)). HSV-1, HSV-2, HCMV and HHV-8 were negative in both MS patients and controls samples. In MS patients, EBV, HHV-7, HHV-6 and VZV were detected in 31.3%, 33.3%, 5.8% and 7.8% of samples, respectively, compared with 3.9%, 9.8%, 1.96% and 1.96%, respectively, of samples from controls. We found a statistically significant difference only for EBV DNA and for HHV-7 DNA prevalence (P < 0.001 and P = 0.03). Although these results indicate lack of apparent association in terms of gender, type of diagnosis, symptoms, disease score and ß interferon treatment between EBV or HHV-7 to MS among Tunisian patients, heterogeneity related to genetic polymorphism as well as geographical distribution of the disease and of pathogens may be of significance.

Re-evaluation of the genome sequence of guinea pig cytomegalovirus.

Congenital infection by human cytomegalovirus (HCMV) is a major cause of birth defects and developmental abnormalities. Since guinea pig cytomegalovirus (GPCMV) crosses the placenta and causes infection in utero, GPCMV models are useful for studies of the mechanisms of transplacental transmission. During our characterization of a genomic locus required for GPCMV dissemination in animals, we found that the nucleotide sequence in and around the nearby immediate-early genes in our lineage of GPCMV strain 22122 [designated GPCMV (ATCC-P5)] showed clear differences from that reported previously for the same strain [designated GPCMV (UMN)] passaged extensively in vitro. Since in vitro passaging of HCMV is known to result in genetic alterations, especially in the UL128-UL131A locus, and loss of growth ability in particular cell types, in this study we determined the complete genome sequence of GPCMV (ATCC-P5), which grows efficiently in animals. A total of 359 differences were identified between the genome sequences of GPCMV (UMN) and GPCMV (ATCC-P5), and these resulted in structural differences in 29 protein-encoding regions. In addition, some genes predicted from our analysis but not from GPCMV (UMN) are well conserved among cytomegaloviruses. An additional 18 passages of GPCMV (ATCC-P5) in vitro generated no further marked alterations in these genes or in the locus corresponding to the HCMV UL128-UL131A. Our analyses indicate that the published sequence of GPCMV (UMN) contains a substantial number of sequencing errors and, possibly, some mutations resulting from a long history of passaging in vitro. Our re-evaluation of the genetic content of GPCMV will provide a solid foundation for future studies.