Concurrent Genotyping and Quantitation of Cytomegalovirus gB Genotypes in Solid-Organ-Transplant Recipients by Use of a Real-Time PCR Assay

Pang, Xiaoli; Humar, Atul; Preiksaitis, Jutta K.

doi:10.1128/jcm.01341-08

Cited by 50 publications

(48 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Further analysis, such as deep sequencing (29) or subcloning of the PCR product amplified from PBMCs obtained from ES patients, is required to confirm mixed populations of the virus in these isolates. Previous studies determined that mixed infections of CMV occurred in 15 to 50% of transplant recipients based on molecular epidemiological analysis of glycoprotein genes (30)(31)(32)(33)(34)(35). Moreover, mixed viral infections have been linked to high viral loads (31,32), delayed viral clearance (31), and higher rates of viral recurrence (31).…”

Section: Discussionmentioning

confidence: 99%

Copy Numbers of Telomeric Repeat Sequences of Human Herpesvirus 6B in Clinical Isolates: Possibility of Mixed Infections

et al. 2014

View full text Add to dashboard Cite

In order to determine whether mixed infections of human herpesvirus 6B (HHV-6B) occur in immunocompetent and immunocompromised individuals, we examined the copy numbers of telomeric repeat sequences (TRS) of clinical isolates. In clinical isolates obtained from patients with exanthem subitum caused by primary HHV-6B infection, PCR products with HHV-6B TRS ranging between 400 and 800 bp were amplified. PCR products of various sizes were amplified in four clinical isolates from druginduced hypersensitivity syndrome (DIHS) patients and 15 isolates from hematopoietic stem cell transplant (HSCT) recipients with HHV-6B reactivation. Based on the sequence analysis of the PCR products, the copy numbers of TRS in DIHS and HSCT patients were between 42 and 82 and 22 and >90, respectively. For two of the HSCT recipients, HHV-6B TRS PCR products of different sizes were detected in several isolates from each patient, which suggests mixed HHV-6B infections. In two of the posttransplant HHV-6B encephalitis patients, the sizes of the TRS nested PCR products amplified from the reactivated virus detected in the central nervous system differed from those of the virus detected in initial isolates from peripheral blood mononuclear cells. Taken together, these results suggest that PCR analysis of TRS copy number is a reliable tool for the discrimination of HHV-6B clinical isolates. Additionally, mixed HHV-6B infections occurred in HSCT recipients, and in some cases, compartmentalization of the HHV-6B strains to the central nervous system versus the blood compartment occurred in posttransplant HHV-6B encephalitis patients. Primary human herpesvirus 6B (HHV-6B) infection presenting as exanthem subitum (ES) (1, 2) is considered a benign febrile illness and rarely causes neurological complications, such as febrile convulsion and encephalitis (3, 4). In addition to the primary infection, HHV-6 reactivation may be associated with acute graftversus-host disease (5-8), graft rejection (9), and encephalitis (10-13) in transplant recipients. Moreover, the virus can be reactivated in patients with drug-induced hypersensitivity syndrome (DIHS), which is a severe form of drug allergy that is characterized by fever, skin rash, lymphadenopathy, hepatitis, and leukocytosis (14-16). Active HHV-6B infection generally occurs only once throughout life (at the time of the primary infection) in immunocompetent individuals, but it may occur several times in transplant recipients (8) or DIHS patients (17). Frequent HHV-6B reactivation, as evidenced by the repeated isolation of the virus during an active viral infection, was observed in hematopoietic stem cell transplant (HSCT) recipients (8, 18).Previous reports detected mixed infections with multiple cytomegalovirus (CMV) strains in the same human herpesvirus subfamily (Betaherpesvirinae subfamily) as HHV-6B in a variety of patient populations, including immunocompetent and immunocompromised patients (19)(20)(21)(22). Furthermore, other studies have suggested that the genotype of the virus may be associate...

show abstract

Section: Discussionmentioning

confidence: 99%

Copy Numbers of Telomeric Repeat Sequences of Human Herpesvirus 6B in Clinical Isolates: Possibility of Mixed Infections

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Mixed populations of CMV genomes within an infected individual have been observed by analyzing gB genotypes (122)(123)(124)(125)(126)(127)(128)(129)(130) as well as the genotypes for additional virion envelope proteins: gN, gO, gH, and gL (127)(128)(129). These differences permitted the categorization of 4 polymorphic gB genotypes (131), 7 polymorphic gO genotypes, and 4 polymorphic gN genotypes (132,133).…”

Section: Strain Polymorphism Contributes To Immune Evasionmentioning

confidence: 99%

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

Gardner

Tortorella

2016

Microbiol Mol Biol Rev

View full text Add to dashboard Cite

SUMMARYThe prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease.

show abstract

“…Even cloning of PCR products and subsequent Sanger sequencing do not allow a sensitive assessment of the genotypes present unless an extremely large number of individual clones is sequenced. Improved genotype-specific real-time-PCR-based assays have been established recently for gB and gH genotyping, and these allow simultaneous detection and quantitation of distinct genotypes in mixed infections down to a level of 5% or even less (14,28).In the present study, we determined the relative amounts of different HCMV genotypes in clinical samples from lung transplant patients by using the highly sensitive ultradeep-pyrosequencing (UDPS) technology. UDPS genotyping of three of the most variable genes within the HCMV genome, the gN, gO, and UL139 genes, revealed that there is a substantially higher diversity of HCMV genotype populations in patients with mixed HCMV infection than has been recognized previously and that the HCMV strains show a distinct pattern in…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Deep Sequencing Reveals Highly Complex Dynamics of Human Cytomegalovirus Genotypes in Transplant Patients over Time

Görzer

Godballe

Trajanoski

et al. 2010

J Virol

111

105

View full text Add to dashboard Cite

In lung transplant patients undergoing immunosuppression, more than one human cytomegalovirus (HCMV) genotype may emerge during follow-up, and this could be critical for the outcome of HCMV infection. Up to now, many cases of infection with multiple HCMV genotypes were probably overlooked due to the limitations of the current genotyping approaches. We have now analyzed mixed-genotype infections in 17 clinical samples from 9 lung transplant patients using the highly sensitive ultradeep-pyrosequencing (UDPS) technology. UDPS genotyping was performed at three variable HCMV genes, coding for glycoprotein N (gN), glycoprotein O (gO), and UL139. Simultaneous analysis of a mean of 10,430 sequence reads per amplicon allowed the relative amounts of distinct genotypes in the samples to be determined down to 0.1% to 1% abundance. Complex mixtures of up to six different HCMV genotypes per sample were observed. In all samples, no more than two major genotypes accounted for at least 88% of the HCMV DNA load, and these were often accompanied by up to four low-abundance genotypes at frequencies of 0.1% to 8.6%. No evidence for the emergence of new genotypes or sequence changes over time was observed. However, analysis of different samples withdrawn from the same patients at different time points revealed that the relative levels of replication of the individual HCMV genotypes changed within a mixed-genotype population upon reemergence of the virus. Our data show for the first time that, similar to what has been hypothesized for the murine model, HCMV reactivation in humans seems to occur stochastically.Human cytomegalovirus (HCMV) is the most important viral pathogen affecting patients after solid organ transplantation (12,18,29). Lung transplant recipients have an especially high risk of acquiring severe and sometimes fatal HCMV infections, which are also associated directly or indirectly with graft rejection and bronchiolitis obliterans syndrome (6, 53).Human cytomegalovirus is a double-stranded DNA virus with a genome size of about 236 kb that is predicted to contain 165 protein-coding genes (9). Although most of the HCMV genome is highly conserved among the various HCMV strains, a subset of genes exhibits a high degree of variability, as has been shown by genome-wide sequence analysis as well as by examination of individual genes. A high level of genetic heterogeneity usually occurs in a limited number of distinct genotypes (9, 32, 33). Among the most variable genes are the viral envelope glycoprotein N (gN) and gO genes and the gene encoding the predicted membrane glycoprotein UL139. Sequence analysis of highly polymorphic regions within these three genes allows the discrimination of seven distinct gN genotypes (30, 31) and eight distinct gO and UL139 genotypes (3,24,35,45). The existence of such a large number of distinct genotypes provides a useful tool for investigating HCMV population diversity within a host.Reinfection with different HCMV strains is possible in the human host, and several strains can accumulate during...

show abstract

Concurrent Genotyping and Quantitation of Cytomegalovirus gB Genotypes in Solid-Organ-Transplant Recipients by Use of a Real-Time PCR Assay

Cited by 50 publications

References 41 publications

Copy Numbers of Telomeric Repeat Sequences of Human Herpesvirus 6B in Clinical Isolates: Possibility of Mixed Infections

Copy Numbers of Telomeric Repeat Sequences of Human Herpesvirus 6B in Clinical Isolates: Possibility of Mixed Infections

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

Deep Sequencing Reveals Highly Complex Dynamics of Human Cytomegalovirus Genotypes in Transplant Patients over Time

Contact Info

Product

Resources

About