Human cytomegalovirus haplotype reconstruction reveals high diversity due to superinfection and evidence of within-host recombination.
ABSTRACT: Recent sequencing efforts have led to estimates of human cytomegalovirus (HCMV) genome-wide intrahost diversity that rival those of persistent RNA viruses [Renzette N, Bhattacharjee B, Jensen JD, Gibson L, Kowalik TF (2011) PLoS Pathog 7:e1001344]. Here, we deep sequence HCMV genomes recovered from single and longitudinally collected blood samples from immunocompromised children to show that the observations of high within-host HCMV nucleotide diversity are explained by the frequent occurrence of mixed infections caused by genetically distant strains. To confirm this finding, we reconstructed within-host viral haplotypes from short-read sequence data. We verify that within-host HCMV nucleotide diversity in unmixed infections is no greater than that of other DNA viruses analyzed by the same sequencing and bioinformatic methods and considerably less than that of human immunodeficiency and hepatitis C viruses. By resolving individual viral haplotypes within patients, we reconstruct the timing, likely origins, and natural history of superinfecting strains. We uncover evidence for within-host recombination between genetically distinct HCMV strains, observing the loss of the parental virus containing the nonrecombinant fragment. The data suggest selection for strains containing the recombinant fragment, generating testable hypotheses about HCMV evolution and pathogenesis. These results highlight that high HCMV diversity present in some samples is caused by coinfection with multiple distinct strains and provide reassurance that within the host diversity for single-strain HCMV infections is no greater than for other herpesviruses.
Project description:Human cytomegalovirus (HCMV) infection is the leading non-genetic cause of congenital birth defects worldwide. While several studies have addressed the genetic composition of viral populations in newborns diagnosed with HCMV, little is known regarding mother-to-child viral transmission dynamics and how therapeutic interventions may impact within-host viral populations. Here, we investigate how preexisting CMV-specific antibodies shape the maternal viral population and intrauterine virus transmission. Specifically, we characterize the genetic composition of CMV populations in a monkey model of congenital CMV infection to examine the effects of passively-infused hyperimmune globulin (HIG) on viral population genetics in both maternal and fetal compartments. In this study, 11 seronegative, pregnant monkeys were challenged with rhesus CMV (RhCMV), including a group pretreated with a standard potency HIG preparation (n = 3), a group pretreated with a high-neutralizing potency HIG preparation (n = 3), and an untreated control group (n = 5). Targeted amplicon deep sequencing of RhCMV glycoprotein B and L genes revealed that one of the three strains present in the viral inoculum (UCD52) dominated maternal and fetal viral populations. We identified minor haplotypes of this strain and characterized their dynamics. Many of the identified haplotypes were consistently detected at multiple timepoints within sampled maternal tissues, as well as across tissue compartments, indicating haplotype persistence over time and transmission between maternal compartments. However, haplotype numbers and diversity levels were not appreciably different between control, standard-potency, and high-potency pretreatment groups. We found that while the presence of maternal antibodies reduced viral load and congenital infection, it had no apparent impact on intrahost viral genetic diversity at the investigated loci. Interestingly, some minor haplotypes present in fetal and maternal-fetal interface tissues were also identified as minor haplotypes in corresponding maternal tissues, providing evidence for a loose RhCMV mother-to-fetus transmission bottleneck even in the presence of preexisting antibodies.
Project description:Wolbachia pipientis, a diverse group of ?-proteobacteria, can alter arthropod host reproduction and confer a reproductive advantage to Wolbachia-infected females (cytoplasmic incompatibility (CI)). This advantage can alter host population genetics because Wolbachia-infected females produce more offspring with their own mitochondrial DNA (mtDNA) haplotypes than uninfected females. Thus, these host haplotypes become common or fixed (selective sweep). Although simulations suggest that for a CI-mediated sweep to occur, there must be a transient phase with repeated initial infections of multiple individual hosts by different Wolbachia strains, this has not been observed empirically. Wolbachia has been found in the tsetse fly, Glossina fuscipes fuscipes, but it is not limited to a single host haplotype, suggesting that CI did not impact its population structure. However, host population genetic differentiation could have been generated if multiple Wolbachia strains interacted in some populations. Here, we investigated Wolbachia genetic variation in G. f. fuscipes populations of known host genetic composition in Uganda. We tested for the presence of multiple Wolbachia strains using Multi-Locus Sequence Typing (MLST) and for an association between geographic region and host mtDNA haplotype using Wolbachia DNA sequence from a variable locus, groEL (heat shock protein 60).MLST demonstrated that some G. f. fuscipes carry Wolbachia strains from two lineages. GroEL revealed high levels of sequence diversity within and between individuals (Haplotype diversity = 0.945). We found Wolbachia associated with 26 host mtDNA haplotypes, an unprecedented result. We observed a geographical association of one Wolbachia lineage with southern host mtDNA haplotypes, but it was non-significant (p = 0.16). Though most Wolbachia-infected host haplotypes were those found in the contact region between host mtDNA groups, this association was non-significant (p = 0.17).High Wolbachia sequence diversity and the association of Wolbachia with multiple host haplotypes suggest that different Wolbachia strains infected G. f. fuscipes multiple times independently. We suggest that these observations reflect a transient phase in Wolbachia evolution that is influenced by the long gestation and low reproductive output of tsetse. Although G. f. fuscipes is superinfected with Wolbachia, our data does not support that bidirectional CI has influenced host genetic diversity in Uganda.
Project description:BACKGROUND:In 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. METHODS:High-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. RESULTS:Among 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. CONCLUSIONS:Breast milk from HIV-infected women contains multiple HCMV strains of high genotypic complexity and thus constitutes a major source for transmitting viral diversity.
Project description:The diversity of RNA viruses dictates their evolution in a particular host, community or environment. Here, we reported within- and between-host pH1N1virus diversity at consensus and sub-consensus levels over a three-year period (2015-2017) and its implications on disease severity. A total of 90 nasal samples positive for the pH1N1 virus were deep-sequenced and analyzed to detect low-frequency variants (LFVs) and haplotypes. Parallel evolution of LFVs was seen in the hemagglutinin (HA) gene across three scales: among patients (33%), across years (22%), and at global scale. Remarkably, investigating the emergence of LFVs at the consensus level demonstrated that within-host virus evolution recapitulates evolutionary dynamics seen at the global scale. Analysis of virus diversity at the HA haplotype level revealed the clustering of low-frequency haplotypes from early 2015 with dominant strains of 2016, indicating rapid haplotype evolution. Haplotype sharing was also noticed in all years, strongly suggesting haplotype transmission among patients infected during a specific influenza season. Finally, more than half of patients with severe symptoms harbored a larger number of haplotypes, mostly in patients under the age of five. Therefore, patient age, haplotype diversity, and the presence of certain LFVs should be considered when interpreting illness severity. In addition to its importance in understanding virus evolution, sub-consensus virus diversity together with whole genome sequencing is essential to explain variabilities in clinical outcomes that cannot be explained by either analysis alone.
Project description:BACKGROUND:Numerous recent studies have shown that resident symbiotic microorganisms of insects play a fundamental role in host ecology and evolution. The lepidopteran pest, African armyworm (Spodoptera exempta), is a highly migratory and destructive species found throughout sub-Saharan Africa, that can experience eruptive outbreaks within the space of a single generation, making predicting population dynamics and pest control forecasting extremely difficult. Three strains of Wolbachia have recently been identified infecting this species in populations sampled from Tanzania. In this study, we examined the interaction between Wolbachia pipiensis infections and the co-inherited marker, mtDNA, within populations of armyworm, as a means to investigate the population biology and evolutionary history of Wolbachia and its host. RESULTS:A Wolbachia-infected isofemale line was established in the laboratory. Phenotypic studies confirmed the strain wExe1 as a male-killer. Partial sequencing of the mitochondrial COI gene from 164 individual field-collected armyworm of known infection status revealed 17 different haplotypes. There was a strong association between Wolbachia infection status and mtDNA haplotype, with a single dominant haplotype, haplo1 (90.2% prevalence), harbouring the endosymbiont. All three Wolbachia strains were associated with this haplotype. This indicates that Wolbachia may be driving a selective sweep on armyworm haplotype diversity. Despite very strong biological and molecular evidence that the samples represent a single species (including from nuclear 28S gene markers), the 17 haplotypes did not fall into a monophyletic clade within the Spodoptera genus; with six haplotypes (2 each from 3 geographically separate populations) differing by >11% in their nucleotide sequence to the other eleven. CONCLUSIONS:This study suggests that three strains of Wolbachia may be driving a selective sweep on armyworm haplotype diversity, and that based on COI sequence data, S. exempta is not a monophyletic group within the Spodoptera genus. This has clear implications for the use of mtDNA as neutral genetic markers in insects, and also demonstrates the impact of Wolbachia infections on host evolutionary genetics.
Project description:A new pathogen strain can penetrate an immune host population only if it can escape immunity generated against the original strain. This model is best understood with influenza viruses, in which genetic drift creates antigenically distinct strains that can spread through host populations despite the presence of immunity against previous strains. Whether this selection model for new strains applies to complex pathogens responsible for endemic persistent infections, such as anaplasmosis, relapsing fever, and sleeping sickness, remains untested. These complex pathogens undergo rapid within-host antigenic variation by using sets of chromosomally encoded variants. Consequently, immunity is developed against a large repertoire of variants, dramatically changing the scope of genetic change needed for a new strain to evade existing immunity and establish coexisting infection, termed strain superinfection. Here, we show that the diversity in the alleles encoding antigenic variants between strains of a highly antigenically variant pathogen was equal to the diversity within strains, reflecting equivalent selection for variants to overcome immunity at the host population level as within an individual host. This diversity among strains resulted in expression of nonoverlapping variants that allowed a new strain to evade immunity and establish superinfection. Furthermore, we demonstrated that a single distinct allele allows strain superinfection. These results indicate that there is strong selective pressure to increase the diversity of the variant repertoire beyond what is needed for persistence within an individual host and provide an explanation, competition at the host population level, for the large genomic commitment to variant gene families in persistent pathogens.
Project description:Infections with human cytomegalovirus (HCMV) are highly prevalent in the general population as the virus has evolved the capacity to undergo distinct replication strategies resulting in lytic, persistent, and latent infections. During the latent life cycle, HCMV resides in subsets of cells within the hematopoietic cell compartment, including hematopoietic progenitor cells (HPCs) and peripheral blood monocytes. Since only a small fraction of these cell types harbor viral genomes during natural latency, identification and analysis of distinct changes mediated by viral infection are difficult to assess. In order to characterize latent infections of HPCs, we used an approach that involves complementation of deficiencies within the human pyrimidine salvage pathway, thus allowing for conversion of labeled uracil into rUTP. Here, we report the development of a recombinant HCMV that complements the defective human pyrimidine salvage pathway, allowing incorporation of thiol containing UTP into all RNA species that are synthesized within an infected cell. This virus grows to wild-type kinetics and can establish a latent infection within two distinct culture models of HCMV latency. Using this recombinant HCMV, we report the specific labeling of transcripts only within infected cells. These transcripts reveal a transcriptional landscape during HCMV latency that is distinct from uninfected cells. The utility of this labeling system allows for the identification of distinct changes within host transcripts and will shed light on characterizing how HCMV establishes and maintains latency.IMPORTANCE HCMV is a significant pathogen that accounts for a substantial amount of complications within the immunosuppressed and immunocompromised. Of particular significance is the capacity of HCMV to reactivate within solid tissue and bone marrow transplant recipients. While it is known that HCMV latency resides within a fraction of HPCs and monocytes, the exact subset of cells that harbor latent viral genomes during natural infections remain uncharacterized. The capacity to identify changes within the host transcriptome during latent infections is critical for developing approaches that therapeutically or physically eliminate latent viral genome containing cells and will represent a major breakthrough for reducing complications due to HCMV reactivation posttransplant. In this report, we describe the generation and use of a recombinant HCMV that allows specific and distinct labeling of RNA species that are produced within virally infected cells. This is a critical first step in identifying how HCMV affects the host cell during latency and more importantly, allows one to characterize cells that harbor latent HCMV.
Project description:Modulation of host DNA synthesis is essential for many viruses to establish productive infections and contributes to viral diseases. Human cytomegalovirus (HCMV), a large DNA virus, blocks host DNA synthesis and deregulates cell cycle progression. We report that pUL117, a viral protein that we recently identified, is required for HCMV to block host DNA synthesis. Mutant viruses in which pUL117 was disrupted, either by frame-shift mutation or by a protein destabilization-based approach, failed to block host DNA synthesis at times after 24 hours post infection in human foreskin fibroblasts. Furthermore, pUL117-deficient virus stimulated quiescent fibroblasts to enter S-phase, demonstrating the intrinsic ability of HCMV to promote host DNA synthesis, which was suppressed by pUL117. We examined key proteins known to be involved in inhibition of host DNA synthesis in HCMV infection, and found that many were unlikely involved in the inhibitory activity of pUL117, including geminin, cyclin A, and viral protein IE2, based on their expression patterns. However, the ability of HCMV to delay the accumulation of the mini-chromosome maintenance (MCM) complex proteins, represented by MCM2 and MCM4, and prevent their loading onto chromatin, was compromised in the absence of pUL117. When expressed alone, pUL117 slowed cell proliferation, delayed DNA synthesis, and inhibited MCM accumulation. Knockdown of MCM proteins by siRNA restored the ability of pUL117-deficient virus to block cellular DNA synthesis. Thus, targeting MCM complex is one mechanism pUL117 employs to help block cellular DNA synthesis during HCMV infection. Our finding substantiates an emerging picture that deregulation of MCM is a conserved strategy for many viruses to prevent host DNA synthesis and helps to elucidate the complex strategy used by a large DNA virus to modulate cellular processes to promote infection and pathogenesis.
Project description:BACKGROUND: The relatively recent introduction of a highly efficient mosquito vector and an avian pathogen (Plasmodium relictum) to an isolated island ecosystem with naïve, highly susceptible avian hosts provides a unique opportunity to investigate evolution of virulence in a natural system. Mixed infections can significantly contribute to the uncertainty in host-pathogen dynamics with direct impacts on virulence. Toward further understanding of how host-parasite and parasite-parasite relationships may impact virulence, this study characterizes within-host diversity of malaria parasite populations based on genetic analysis of the trap (thrombospondin-related anonymous protein) gene in isolates originating from Hawaii, Maui and Kauai Islands. METHODS: A total of 397 clones were produced by nested PCR amplification and cloning of a 1664 bp fragment of the trap gene from two malarial isolates, K1 (Kauai) and KV115 (Hawaii) that have been used for experimental studies, and from additional isolates from wild birds on Kauai, Maui and Hawaii Islands. Diversity of clones was evaluated initially by RFLP-based screening, followed by complete sequencing of 33 selected clones. RESULTS: RFLP analysis of trap revealed a minimum of 28 distinct RFLP haplotypes among the 397 clones from 18 birds. Multiple trap haplotypes were detected in every bird evaluated, with an average of 5.9 haplotypes per bird. Overall diversity did not differ between the experimental isolates, however, a greater number of unique haplotypes were detected in K1 than in KV115. We detected high levels of clonal diversity with clear delineation between isolates K1 and KV115 in a haplotype network. The patterns of within-host haplotype clustering are consistent with the possibility of a clonal genetic structure and rapid within-host mutation after infection. CONCLUSION: Avian malaria (P. relictum) and Avipoxvirus are the significant infectious diseases currently affecting the native Hawaiian avifauna. This study shows that clonal diversity of Hawaiian isolates of P. relictum is much higher than previously recognized. Mixed infections can significantly contribute to the uncertainty in host-pathogen dynamics with direct implications for host demographics, disease management strategies, and evolution of virulence. The results of this study indicate a widespread presence of multiple-genotype malaria infections with high clonal diversity in native birds of Hawaii, which when coupled with concurrent infection with Avipoxvirus, may significantly influence evolution of virulence.
Project description:Human cytomegalovirus (HCMV) has a relatively large and complex genome, a protracted lytic replication cycle, and employs a strategy of replicational latency as part of its lifelong persistence in the infected host. An important form of gene regulation in plants and animals revolves around a type of small RNA known as microRNA (miRNA). miRNAs can serve as major regulators of key developmental pathways, as well as provide subtle forms of regulatory control. The human genome encodes over 900 miRNAs, and miRNAs are also encoded by some viruses, including HCMV, which encodes at least 14 miRNAs. Some of the HCMV miRNAs are known to target both viral and cellular genes, including important immunomodulators. In addition to expressing their own miRNAs, infections with some viruses, including HCMV, can result in changes in the expression of cellular miRNAs that benefit virus replication. In this review, we summarize the connections between miRNAs and HCMV biology. We describe the nature of miRNA genes, miRNA biogenesis and modes of action, methods for studying miRNAs, HCMV-encoded miRNAs, effects of HCMV infection on cellular miRNA expression, roles of miRNAs in HCMV biology, and possible HCMV-related diagnostic and therapeutic applications of miRNAs.