The major obstacle to curing HIV infection is the persistence of cells with intact proviruses that can produce replication-competent virus. This HIV reservoir is believed to exist primarily in CD4+ ...T-cells and is stable despite years of suppressive antiretroviral therapy. A potential mechanism for HIV persistence is clonal expansion of infected cells, but how often such clones carry replication-competent proviruses has been controversial. Here, we used single-genome sequencing to probe for identical HIV sequence matches among viruses recovered in different viral outgrowth cultures and between the sequences of outgrowth viruses and proviral or intracellular HIV RNA sequences in uncultured blood mononuclear cells from eight donors on suppressive ART with diverse proviral populations. All eight donors had viral outgrowth virus that was fully susceptible to their current ART drug regimen. Six of eight donors studied had identical near full-length HIV RNA sequences recovered from different viral outgrowth cultures, and one of the two remaining donors had identical partial viral sequence matches between outgrowth virus and intracellular HIV RNA. These findings provide evidence that clonal expansion of HIV-infected cells is an important mechanism of reservoir persistence that should be targeted to cure HIV infection.
To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during ART, we looked for evidence of viral replication in 5 donors after up to 13 years of viral ...suppression. We characterized proviral populations in lymph nodes and peripheral blood before and during ART, evaluated the levels of viral RNA expression in single lymph node and blood cells, and characterized the proviral integration sites in paired lymph node and blood samples. Proviruses with identical sequences, identical integration sites, and similar levels of RNA expression were found in lymph nodes and blood samples collected during ART, and no single sequence with significant divergence from the pretherapy population was present in either blood or lymph nodes. These findings show that all detectable persistent HIV-1 infection is consistent with maintenance in lymph nodes by clonal proliferation of cells infected before ART and not by ongoing viral replication during ART.
Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information ...regarding the structure of persistent HIV DNA populations; however, until recently, there was noway to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for “viral reconstruction” to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality.
BACKGROUNDHIV-1 viremia that is not suppressed by combination antiretroviral therapy (ART) is generally attributed to incomplete medication adherence and/or drug resistance. We evaluated individuals ...referred by clinicians for nonsuppressible viremia (plasma HIV-1 RNA above 40 copies/mL) despite reported adherence to ART and the absence of drug resistance to the current ART regimen.METHODSSamples were collected from at least 2 time points from 8 donors who had nonsuppressible viremia for more than 6 months. Single templates of HIV-1 RNA obtained from plasma and viral outgrowth of cultured cells and from proviral DNA were amplified by PCR and sequenced for evidence of clones of cells that produced infectious viruses. Clones were confirmed by host-proviral integration site analysis.RESULTSHIV-1 genomic RNA with identical sequences were identified in plasma samples from all 8 donors. The identical viral RNA sequences did not change over time and did not evolve resistance to the ART regimen. In 4 of the donors, viral RNA sequences obtained from plasma matched those sequences from viral outgrowth cultures, indicating that the viruses were replication competent. Integration sites for infectious proviruses from those 4 donors were mapped to the introns of the MATR3, ZNF268, ZNF721/ABCA11P, and ABCA11P genes. The sizes of the clones were estimated to be from 50 million to 350 million cells.CONCLUSIONThese findings show that clones of HIV-1-infected cells producing virus can cause failure of ART to suppress viremia. The mechanisms involved in clonal expansion and persistence need to be defined to effectively target viremia and the HIV-1 reservoir.FUNDINGNational Cancer Institute, NIH; Howard Hughes Medical Research Fellows Program, Howard Hughes Medical Institute; Bill and Melinda Gates Foundation; Office of AIDS Research; American Cancer Society; National Cancer Institute through a Leidos subcontract; National Institute for Allergy and Infectious Diseases, NIH, to the I4C Martin Delaney Collaboratory; University of Rochester Center for AIDS Research and University of Rochester HIV/AIDS Clinical Trials Unit.
Surveys of microbial populations in environmental niches of interest often utilize sequence variation in the gene encoding the ribosomal small subunit (the 16S rRNA gene). Generally, these surveys ...target the 16S genes using semi-degenerate primers to amplify portions of a subset of bacterial species, sequence the amplicons in bulk, and assign to putative taxonomic categories by comparison to databases purporting to connect specific sequences in the main variable regions of the gene to specific organisms. Due to sequence length constraints of the most popular bulk sequencing platforms, the primers selected amplify one to three of the nine variable regions, and taxonomic assignment is based on relatively short stretches of sequence (150-500 bases). We demonstrate that taxonomic assignment is improved through reduced unassigned reads by including a survey of near-full-length sequences specific to the target environment, using a niche of interest represented by the upper respiratory tract (URT) of cattle. We created a custom Bovine URT database from these longer sequences for assignment of shorter, less expensive reads in comparisons of the upper respiratory tract among individual animals. This process improves the ability to detect changes in the microbial populations of a given environment, and the accuracy of defining the content of that environment at increasingly higher taxonomic resolution.
Abstract
The U.S. Meat Animal Research Center was the first entity in the United States to import the Romanov breed and it has been maintained as a closed flock for over 30 yr. Incorporating this ...super-prolific breed into crossbred and composite populations has resulted in large improvements in ewe productivity. However, few have quantified factors contributing to genetic and nongenetic variation in ewe reproduction and lamb growth within purebred Romanov populations, which were the objectives of this study. The pedigree contained a total of 8,683 lambs born to 218 and 1,600 unique sires and dams, respectively. Number of lambs born on a per ewe exposed (NLBE) and lambing (NLBL) basis were analyzed in univariate repeatability animal models. As expected, the proportion of phenotypic variance (σP2) in litter size attributable to additive genetic (0.06 to 0.08) and permanent environmental (0.05 to 0.07) effects of the ewe was low. The service sire permanent environmental effect contributed to a small but significant amount of σP2 in NLBE (0.03) but not NLBL. However, the service sire additive genetic effect did not influence σP2 in NLBE or NLBL. Lamb body weight was recorded at birth (BWB) and upon weaning from either milk replacer (~30 d; BWW-N) or their dam (~60 d; BWW-D) and were analyzed in a three-trait model with random additive direct and maternal effects. Estimated direct heritabilities were low for all body weight (BW) traits (0.07 to 0.10). Maternal heritability was moderate for BWB (0.34) but low for weaning BW (0.11 to 0.18). This was the first to report direct and maternal genetic correlations between BW of nursery- and dam-reared lambs, and both were estimated to be moderate (0.43 to 0.47). Additionally, the direct and maternal effects of BWB were more strongly correlated with BWW-N (0.74 to 0.82) than BWW-D (0.17 to 0.33). Despite inbreeding coefficients having increased at a rate of 0.33% per birth year (1986 to 2019) in this flock, they were not consistently associated with reductions in ewe or lamb performance. Parameter estimates generally agreed with those from less-prolific breeds, and results indicate that selection can be an effective means of improving subcomponents of ewe productivity.
Abstract
Genotyping pools of commercial cattle and individual seedstock animals may reveal hidden relationships between sectors enabling use of commercial data for genetic evaluation. However, ...commercial data capture may be compromised by inexact pool formation. We aimed to estimate the concordance between distances or genomic covariance among pooling allele frequencies (PAFs) of DNA pools comprised of 100 animals with 0% or 50% overlap of animals in common between pools. Cattle lung samples were collected from a commercial beef processing plant on a single day. Six pools of 100 animals each were constructed so that overlap between pools was 0% or 50%. Two pools of all 200 animals were constructed to estimate PAFs for all 200 animals. Frozen lung tissue (0.01 g) from each animal was weighed into a tube containing a pool; there were two pools of 200 animals each and six pools of 100 animals each. Every contribution of an individual animal was an independent measurement to insure independence of pooling errors. Lung samples were kept on dried ice during the pooling process to keep them from thawing. The eight pools were then assayed for approximately 100,000 single nucleotide polymorphisms (SNP). PAF for each SNP and pool was based on the relative intensity of the two dyes used to detect the alleles rather than genotype calls which are not tractable from pooling data. Euclidean distances and genomic relationships among the PAFs for the eight pools were estimated and distances were tested for concordance with pool overlap using permutation-based analysis of distance. Distances among pools were concordant with the planned overlap of animals shared between pools (P = 0.0024); pool overlap accounted for 70% of the variation and pooling error accounted for 30%. Pools containing 100 animals with no overlap were the most distant from one another and pools with 50% overlap were the least distant. This work shows that we can discern differences in distance between pairs of overlapping DNA pools sharing 0% and 50% of the animals. Genomic correlations among nonoverlapping pools indicated that nonoverlapping pool pairs did not share many related animals because genomic correlations were near zero for these pairs. On the other hand, one pair of nonoverlapping pools likely contained related animals between pools because the correlation was 0.21. Pools sharing 50% overlap ranged in genomic relationship between 0.21 and 0.39 (N = 12).
Relevance and accuracy of genetic evaluations of bulls produced in the seedstock sector can be improved through capturing commercial data from ranches, feedlots, and processing plants. We provide evidence that overlapping DNA pools provides a cost-effective strategy to identify genetic connections between seedstock and commercial sectors of the cattle industry despite pooling error.
Lay Summary
Genetic evaluation of seedstock cattle could benefit from commercial data. There are hidden relationships between commercial and seedstock sectors because many commercial producers buy bulls from the seedstock sector. Relationships are hidden because pedigree is not tracked in commercial populations. Single nucleotide polymorphism genotypes could reveal these hidden relationships; however, genotyping can be cost prohibitive. Cost of commercial data capture could be decreased by pooling DNA which is a method to genotype groups of animals to use their data in genetic evaluation; however, error from inexact pool formation can complicate interpretation. Results from pools of overlapping random unrelated animals mimic the results from pools sharing relatives with the same degree of shared genomes. For example, a pool of progeny and a pool of the dams of the pooled progeny would produce the same result as two pools sharing 50% overlap of random unrelated animals. We can estimate the relatedness between unknown pools even in the presence of pooling error if an unknown pool comparison is similar to an overlapping pool comparison. Knowing the relationship between seedstock cattle and pools of commercial cattle may allow commercial data to enhance genetic evaluation of seedstock animals.
Understanding the origin of HIV variants during viral rebound may provide insight into the composition of the HIV reservoir and has implications for the design of curative interventions. HIV ...single-genome sequences were obtained from 10 AIDS Clinical Trials Group participants who underwent analytic antiretroviral therapy (ART) interruption (ATI). Rebounding variants were compared with those in pre-ART plasma in all 10 participants and with on-ART peripheral blood mononuclear cell (PBMC)-associated DNA and RNA (CA-RNA) in 7/10 participants. The highest viral diversities were found in the DNA and CA-RNA populations. In 3 of 7 participants, we detected multiple, identical DNA and CA-RNA sequences during suppression on ART that exactly matched plasma HIV sequences. Hypermutated DNA and CA-RNA were detected in four participants, contributing to diversities in these compartments that were higher than in the pre-ART and post-ATI plasma. Shifts in the viral rebound populations could be detected in some participants over the 2- to 3-month observation period. These findings suggest that a source of initial rebound viremia could be populations of infected cells that clonally expanded prior to and/or during ART, some of which were already expressing HIV RNA before treatment was interrupted. These clonally expanding populations of HIV-infected cells may represent an important target for strategies aimed at achieving reservoir reduction and sustained virologic remission.
Antiretroviral therapy alone cannot eradicate the HIV reservoir, and viral rebound is generally rapid after treatment interruption. It has been suggested that clonal expansion of HIV-infected cells is an important mechanism of HIV reservoir persistence, but the contribution of these clonally proliferating cells to the rebounding virus is unknown. We report a study of AIDS Clinical Trials Group participants who underwent treatment interruption and compared rebounding plasma virus with that found within cells prior to treatment interruption. We found several incidences in which plasma HIV variants exactly matched that of multiple proviral DNA copies from infected blood cells sampled before treatment interruption. In addition, we found that these cells were not dormant but were generating unspliced RNA transcripts before treatment was interrupted. Identification of the HIV reservoir and determining its mechanisms for persistence may aid in the development of strategies toward a cure for HIV. (This study was presented in part at the Conference on Retroviruses and Opportunistic Infections, Seattle, WA, February 23 to 26 2015.).
Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here, we describe the first systematic and genome-wide analysis of copy number variations (CNVs) ...in modern domesticated cattle using array comparative genomic hybridization (array CGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH). The array CGH panel included 90 animals from 11 Bos taurus, three Bos indicus, and three composite breeds for beef, dairy, or dual purpose. We identified over 200 candidate CNV regions (CNVRs) in total and 177 within known chromosomes, which harbor or are adjacent to gains or losses. These 177 high-confidence CNVRs cover 28.1 megabases or ;1.07% of the genome. Over 50% of the CNVRs (89/177) were found in multiple animals or breeds and analysis revealed breed-specific frequency differences and reflected aspects of the known ancestry of these cattle breeds. Selected CNVs were further validated by independent methods using qPCR and FISH. Approximately 67% of the CNVRs (119/177) completely or partially span cattle genes and 61% of the CNVRs (108/177) directly overlap with segmental duplications. The CNVRs span about 400 annotated cattle genes that are significantly enriched for specific biological functions, such as immunity, lactation, reproduction, and rumination. Multiple gene families, including ULBP, have gone through ruminant lineage-specific gene amplification. We detected and confirmed marked differences in their CNV frequencies across diverse breeds, indicating that some cattle CNVs are likely to arise independently in breeds and contribute to breed differences. Our results provide a valuable resource beyond microsatellites and single nucleotide polymorphisms to explore the full dimension of genetic variability for future cattle genomic research.
Abstract
We hypothesized cattle that differed in BW gain had different digestive tract microbiota. Two experiments were conducted. In both experiments, steers received a diet that consisted of 8.0% ...chopped alfalfa hay, 20% wet distillers grain with solubles, 67.75% dry-rolled corn, and 4.25% vitamin/mineral mix (including monensin) on a dry matter basis. Steers had ad libitum access to feed and water. In experiment 1, 144 steers (age = 310 ± 1.5 d; BW = 503 ± 37.2 kg) were individually fed for 105 d. Ruminal digesta samples were collected from eight steers with the greatest (1.96 ± 0.02 kg/d) and eight steers with the least ADG (1.57 ± 0.02 kg/d) that were within ±0.32 SD of the mean (10.1 ± 0.05 kg/d) dry matter. In experiment 2, 66 steers (age = 396 ± 1 d; BW = 456 ± 5 kg) were individually fed for 84 d. Rumen, duodenum, jejunum, ileum, cecum, and colon digesta samples were collected from eight steers with the greatest (2.39 ± 0.06 kg/d) and eight steers with the least ADG (1.85 ± 0.06 kg/d) that were within ±0.55 SD of the mean dry matter intake (11.9 ± 0.1 kg/d). In both studies, DNA was isolated and the V1 to V3 regions of the 16S rRNA gene were sequenced. Operational taxonomic units were classified using 0.03 dissimilarity and identified using the Greengenes 16S rRNA gene database. In experiment 1, there were no differences in the Chao1, Shannon, Simpson, and InvSimpson diversity indexes or the permutation multivariate analysis of variance (PERMANOVA; P = 0.57). The hierarchical test returned six clades as being differentially abundant between steer classifications (P < 0.05). In experiment 2, Chao1, Shannon, Simpson, and InvSimpson diversity indexes and PERMANOVA between steer classified as less or greater ADG did not differ (P > 0.05) for the rumen, duodenum, ileum, cecum, and colon. In the jejunum, there tended to be a difference in the Chao1 (P = 0.09) and Simpson diversity (P = 0.09) indexes between steer classifications, but there was no difference in the Shannon (P = 0.14) and InvSimpson (P = 0.14) diversity indexes. Classification groups for the jejunum differed (P = 0.006) in the PERMANOVA. The hierarchical dependence false discovery rate procedure returned 11 clades as being differentially abundant between steer classifications in the jejunum (P < 0.05). The majority of the OTU were in the Families Corynebacteriaceae and Coriobacteriaceae. This study suggests that intestinal differences in the microbiota of ruminants may be associated with animal performance.
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