Sequence evaluation and comparative analysis of novel assays for intact proviral HIV-1 DNA

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon ART-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The Intact Proviral DNA Assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR), is a lower-throughput assay that uses limiting dilution long distance PCR amplification followed by qPCR and near-full length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size. IMPORTANCE The Intact Proviral DNA Assay (IPDA) and Quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 ART-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.

Sequence evaluation and comparative analysis of novel assays for intact proviral HIV-1 DNA

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon ART-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The Intact Proviral DNA Assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR), is a lower-throughput assay that uses limiting dilution long distance PCR amplification followed by qPCR and near-full length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size.ImportanceThe Intact Proviral DNA Assay (IPDA) and Quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 ART-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.

Novel Mammalian Herpesviruses and Lineages within the Gammaherpesvirinae: Cospeciation and Interspecies Transfer

ABSTRACT Novel members of the subfamily Gammaherpesvirinae, hosted by eight mammalian species from six orders (Primates, Artiodactyla, Perissodactyla, Carnivora, Scandentia, and Eulipotyphla), were discovered using PCR with pan-herpesvirus DNA polymerase (DPOL) gene primers and genus-specific glycoprotein B (gB) gene primers. The gB and DPOL sequences of each virus species were connected by long-distance PCR, and contiguous sequences of approximately 3.4 kbp were compiled. Six additional gammaherpesviruses from four mammalian host orders (Artiodactyla, Perissodactyla, Primates, and Proboscidea), for which only short DPOL sequences were known, were analyzed in the same manner. Together with available corresponding sequences for 31 other gammaherpesviruses, alignments of encoded amino acid sequences were made and used for phylogenetic analyses by maximum-likelihood and Bayesian Monte Carlo Markov chain methods to derive a tree which contained two major loci of unresolved branching details. The tree was rooted by parallel analyses that included alpha- and betaherpesvirus sequences. This gammaherpesvirus tree contains 11 major lineages and presents the widest view to date of phylogenetic relationships in any subfamily of the Herpesviridae, as well as the most complex in the number of deep lineages. The tree's branching pattern can be interpreted only in part in terms of the cospeciation of virus and host lineages, and a substantial incidence of the interspecies transfer of viruses must also be invoked.