Multiple Human Cytomegalovirus Strains Research Articles

Human cytomegalovirus strain diversity and dynamics reveal the donor lung as a major contributor after transplantation.

Mixed human cytomegalovirus (HCMV) strain infections are frequent in lung transplant recipients (LTRs). To date, the influence of the donor (D) and recipient (R) HCMV serostatus on intra-host HCMV strain composition and viral population dynamics after transplantation is only poorly understood. Here, we investigated ten pre-transplant lungs from HCMV-seropositive donors and 163 sequential HCMV-DNA-positive plasma and bronchoalveolar lavage samples from fifty LTRs with multiviremic episodes post-transplantation. The study cohort included D+R+ (38 per cent), D+R− (36 per cent), and D−R+ (26 per cent) patients. All samples were subjected to quantitative genotyping by short amplicon deep sequencing, and twenty-four of them were additionally PacBio long-read sequenced for genotype linkages. We find that D+R+ patients show a significantly elevated intra-host strain diversity compared to D+R− and D−R+ patients (P = 0.0089). Both D+ patient groups display significantly higher viral population dynamics than D− patients (P = 0.0061). Five out of ten pre-transplant donor lungs were HCMV DNA positive, whereof three multiple HCMV strains were detected, indicating that multi-strain transmission via lung transplantation is likely. Using long reads, we show that intra-host haplotypes can share distinctly linked genotypes, which limits overall intra-host diversity in mixed infections. Together, our findings demonstrate donor-derived strains as the main source of increased HCMV strain diversity and dynamics post-transplantation. These results foster strategies to mitigate the potential transmission of the donor strain reservoir to the allograft, such as ex vivo delivery of HCMV-selective immunotoxins prior to transplantation to reduce latent HCMV.

Whole-Genome Approach to Assessing Human Cytomegalovirus Dynamics in Transplant Patients Undergoing Antiviral Therapy.

Human cytomegalovirus (HCMV) is the most frequent cause of opportunistic viral infection following transplantation. Viral factors of potential clinical importance include the selection of mutants resistant to antiviral drugs and the occurrence of infections involving multiple HCMV strains. These factors are typically addressed by analyzing relevant HCMV genes by PCR and Sanger sequencing, which involves independent assays of limited sensitivity. To assess the dynamics of viral populations with high sensitivity, we applied high-throughput sequencing coupled with HCMV-adapted target enrichment to samples collected longitudinally from 11 transplant recipients (solid organ, n = 9, and allogeneic hematopoietic stem cell, n = 2). Only the latter presented multiple-strain infections. Four cases presented resistance mutations (n = 6), two (A594V and L595S) at high (100%) and four (V715M, V781I, A809V, and T838A) at low (<25%) frequency. One allogeneic hematopoietic stem cell transplant recipient presented up to four resistance mutations, each at low frequency. The use of high-throughput sequencing to monitor mutations and strain composition in people at risk of HCMV disease is of potential value in helping clinicians implement the most appropriate therapy.

Multiple-Strain Infections of Human Cytomegalovirus With High Genomic Diversity Are Common in Breast Milk From Human Immunodeficiency Virus-Infected Women in Zambia.

BackgroundIn developed countries, human cytomegalovirus (HCMV) is a major pathogen in congenitally infected and immunocompromised individuals, where multiple-strain infection appears linked to disease severity. The situation is less documented in developing countries. In Zambia, breast milk is a key route for transmitting HCMV and carries higher viral loads in human immunodeficiency virus (HIV)–infected women. We investigated HCMV strain diversity.MethodsHigh-throughput sequence datasets were generated from 28 HCMV-positive breast milk samples donated by 22 mothers (15 HIV-infected and 7 HIV-negative) at 4–16 weeks postpartum, then analyzed by genome assembly and novel motif-based genotyping in 12 hypervariable HCMV genes.ResultsAmong the 20 samples from 14 donors (13 HIV-infected and one HIV-negative) who yielded data meeting quality thresholds, 89 of the possible 109 genotypes were detected, and multiple-strain infections involving up to 5 strains per person were apparent in 9 HIV-infected women. Strain diversity was extensive among individuals but conserved compartmentally and longitudinally within them. Genotypic linkage was maintained within hypervariable UL73/UL74 and RL12/RL13/UL1 loci for virus entry and immunomodulation, but not between genes more distant from each other.ConclusionsBreast milk from HIV-infected women contains multiple HCMV strains of high genotypic complexity and thus constitutes a major source for transmitting viral diversity.

Expression Levels of Glycoprotein O (gO) Vary between Strains of Human Cytomegalovirus, Influencing the Assembly of gH/gL Complexes and Virion Infectivity.

The tropism of human cytomegalovirus (HCMV) is influenced by the envelope glycoprotein complexes gH/gL/gO and gH/gL/UL128-131. During virion assembly, gO and the UL128-131 proteins compete for binding to gH/gL in the endoplasmic reticulum (ER). This assembly process clearly differs among strains, since Merlin (ME) virions contain abundant gH/gL/UL128-131 and little gH/gL/gO, whereas TR contains much higher levels of total gH/gL, mostly in the form of gH/gL/gO, but much lower levels of gH/gL/UL128-131 than ME. Remaining questions include (i) what are the mechanisms behind these assembly differences, and (ii) do differences reflect in vitro culture adaptations or natural genetic variations? Since the UL74(gO) open reading frame (ORF) differs in 25% of amino acids between TR and ME, we analyzed recombinant viruses in which the UL74(gO) ORF was swapped. TR virions were >40-fold more infectious than ME. Transcriptional repression of UL128-131 enhanced the infectivity of ME to the level of TR, despite still far lower levels of gH/gL/gO. Swapping the UL74(gO) ORF had no effect on either TR or ME. A quantitative immunoprecipitation approach revealed that gH/gL expression levels were within 4-fold between TR and ME, but the gO expression level was 20-fold lower for ME, which suggested differences in mRNA transcription, translation, or rapid ER-associated degradation of gO. trans-Complementation of gO expression during ME replication gave a 6-fold enhancement of infectivity beyond the 40-fold effect of UL128-131 repression alone. Overall, strain variations in the assembly of gH/gL complexes result from differences in the expression of gO and UL128-131, and selective advantages for reduced UL128-131 expression during fibroblast propagation are much stronger than those for higher gO expression.IMPORTANCE Specific genetic differences between independently isolated HCMV strains may result from purifying selection on de novo mutations arising during propagation in culture or random sampling among the diversity of genotypes present in clinical specimens. Results presented indicate that while reduced UL128-131 expression may confer a powerful selective advantage during cell-free propagation of HCMV in fibroblast cultures, selective pressures for increased gO expression are much weaker. Thus, variation in gO expression among independent strains may represent natural genotype variability present in vivo This may have important implications for virus-host interactions, such as immune recognition, and underscores the value of studying molecular mechanisms of replication using multiple HCMV strains.

Human cytomegalovirus glycoprotein polymorphisms and increasing viral load in AIDS patients

BackgroundMultiple strains infection of human cytomegalovirus (HCMV) was found to be correlated with increased viral load in immunodeficient patients. However, the pathogenic mechanism underlying this correlation remains unclear. To evaluate genetic polymorphisms of HCMV glycoprotein and their potential role in its viral load, HCMV glycoprotein B, N, and O (gB, gN and gO) genotypes was studied in the population of HCMV infected acquired immune deficiency syndrome (AIDS) patients. The association between glycoprotein polymorphisms and HCMV viral load was analyzed.MethodsThe genetic polymorphisms of glycoprotein from sera of 60 HCMV infected AIDS patients was investigated by multiplex nested PCR and sequencing. HCMV viral load was evaluated by quantitative PCR.ResultsgB1, gO1a, and gN4a were the predominant glycoprotein genotypes in HCMV infected AIDS patients and composed 86.96%, 78.8%, and 49.2%, respectively. Only gN4a genotype infection significantly increased viral load (P = 0.048). 71% (43/60) of HCMV infected AIDS patients were found to carry multiple HCMV strains infection. A novel potential linkage of gO1a/gN4a was identified from multiple HCMV infected patients. It was the most frequent occurrence, accounted for 51.5% in 33 patients with gO and gN genotypes infection. Furthermore, the gO1a/gN4a linkage was correlated to an increased viral load (P = 0.020).ConclusionThe gN4a correlates to higher level HCMV load in AIDS patients. Interestingly, a novel gO1a/gN4a linkage is identified from the patients with multiple HCMV strains infection and is also associated with an increased viral load. Therefore, the pathogenic mechanism underlying glycoprotein polymorphisms and interaction of variants should be analyzed further.

Mixed infections with distinct cytomegalovirus glycoprotein B genotypes in Polish pregnant women, fetuses, and newborns

The purpose of this investigation was to describe a distribution of cytomegalovirus (CMV) single and multiple genotypes among infected pregnant women, their fetuses, and newborns coming from Central Poland, as well as congenital cytomegaly outcome. The study involved 278 CMV-seropositive pregnant women, of whom 192 were tested for viral DNAemia. Human cytomegalovirus (HCMV) genotyping was performed for 18 of 34 pregnant women carrying the viral DNA and for 12 of their 15 offspring with confirmed HCMV infections. Anti-HCMV antibodies levels were assessed by chemiluminescence immunoassay (CLIA) and enzyme-linked fluorescence assay (ELFA) tests. Viral DNA loads and genotypes were determined by real-time polymerase chain reaction (PCR) assays for the UL55 gene. In the pregnant women, we identified HCMV gB1, gB2, gB3, and gB4 genotypes. Single gB2, gB3, or gB4 genotypes were observed in 14 (77.8 %) women, while multiple gB1–gB2 or gB2–gB3 genotypes were observed in four (22.2 %). Maternal HCMV genotypes determined the genotypes identified in their fetuses and newborns (p ≤ 0.050). Half of them were infected with single HCMV gB1, gB2, or gB3 genotypes and the other half with multiple gB1–gB2 or gB2–gB3 genotypes. Single and multiple genotypes were observed in both asymptomatic and symptomatic congenital cytomegaly, although no gB3 genotype was identified among asymptomatic cases. In Central Poland, infections with single and multiple HCMV strains occur in pregnant women, as well as in their fetuses and neonates, with both asymptomatic and symptomatic infections. HCMV infections identified in mothers seem to be associated with the viral genotypes in their children.

Human Cytomegalovirus: Bacterial Artificial Chromosome (BAC) Cloning and Genetic Manipulation

The understanding of human cytomegalovirus (HCMV) biology was long hindered by the inability to perform efficient viral genetic analysis. This hurdle was recently overcome when the genomes of multiple HCMV strains were cloned as infectious bacterial artificial chromosomes (BACs). The BAC system takes advantage of the single-copy F plasmid of E. coli that can stably carry large pieces of foreign DNA. In this system, a recombinant HCMV virus carrying a modified F plasmid is first generated in eukaryotic cells. Recombinant viral genomes are then isolated and recovered in E. coli as BAC clones. BAC-captured viral genomes can be manipulated using prokaryotic genetics, and recombinant virus can be reconstituted from BAC transfection in eukaryotic cells. The BAC reverse genetic system provides a reliable and efficient method to introduce genetic alterations into the viral genome in E.coli and subsequently analyze their effects on virus biology in eukaryotic cells.

Stability of human cytomegalovirus genotypes in persistently infected renal transplant recipients

Although most genes are highly conserved among strains of human cytomegalovirus (HCMV), several are unusually variable. By analyzing the sequence of two variable genes (UL146 and UL74) amplified directly from whole blood DNA extracts, multiple HCMV strains were detected in blood samples from 5/11 virus-infected renal transplant patients. These five patients were seronegative prior to transplantation and their likely acquisition of the virus was via the donated organ; HCMV-positive immunocompetent donors may thus be capable of harboring and transmitting multiple virus genotypes. In sequential samples taken up to 140 days post transplant no mutations in either gene were detected from 9/10 patients, and in the remaining patient a single nucleotide change was detected in UL146, and none in UL74. All sequences grouped into previously defined genotypes, with the detection of multiple members of the UL74 group 5 genotype establishing this previously tentative genotype. Additionally, identical sequences were identified in viruses from different patients, including examples from geographically distinct regions. Thus, although UL146 and UL74 exhibit impressive variation among strains, their sequences are maintained stably within and between infected individuals, suggesting that sequence differences between genotypes may be driven by differing gene function.

Simultaneous infection of healthy people with multiple human cytomegalovirus strains

Surface adsorbed chitosan-loaded solid self-nanoemulsifying drug delivery system (S-SNEDDS) of glibenclamide (GBN) was developed. Low cost coffee husk was investigated as a biosorbent for solidification of liquid SNEDDS. Different liquid and S-SNEDDS of GBN were developed by aqueous phase titration method. Developed formulations were characterized in terms of thermodynamic stability, self-nanoemulsification efficiency, droplet size, viscosity, % transmittance, drug content and droplet morphology. Finally, selected liquid and surface adsorbed S-SNEDDS were subjected to in vitro dissolution. In vitro drug release studies showed 98.91% release of GBN from optimized formulation. The release pattern of GBN from S-SNEDDS was found to be sustained release type as compared to immediate release pattern of liquid SNEDDS. The results of solubility studies showed 1492 fold enhancement in GBN solubility in optimized SNEDDS. These results indicate that developed surface adsorbed chitosan loaded S-SNEDDS could be successfully used as a unit dosage form of GBN.

Simultaneous infection of healthy people with multiple human cytomegalovirus strains

Simultaneous infection of healthy people with multiple human cytomegalovirus strains

Coinfection of the immunocompromised but not the immunocompetent host by multiple human cytomegalovirus strains.

Coinfection by multiple human cytomegalovirus (HCMV) strains was investigated in immunocompetent individuals and AIDS patients. Thirty HCMV maternal and fetal or newborn isolate pairs from 9 cases of congenital HCMV infection as well as 36 HCMV isolates and 2 PCR-HCMV-positive CSF samples from 13 AIDS patients were tested by restriction fragment length polymorphism analysis of multiple genome regions. Results from the group of congenital infections showed that: i) all the 9 women with primary HCMV infection presumably harboured a single HCMV strain; ii) all strains were genetically unrelated; iii) isolates from infected fetuses or newborns consisted of a single strain apparently indistinguishable from the maternal strain; iv) no strain variations were observed in isolates from different body sites or in sequential isolates from newborns up to 8 months after birth. Results from the AIDS patient group demonstrated that; i) all patients were infected by unrelated strains; ii) 6/13 (46.1%) patients were coinfected by 2 or more HCMV strains; iii) in a single patient two different HCMV strains were detected in blood and urine, respectively, whereas a mixture of the two was found in the pharynx; iv) 4 patients showed the sequential appearance of a mixed virus population or different strains suggesting sequential reinfections.