Phylogenetic tree of orthopoxviruses including ERPV.

<p>Concatenated sequences of 96 ORFs conserved in each viral genome were used to perform the analysis. ERPV branches from the same node as ECTV.</p

Comparison of genomes of ERPV and ECTV-Nav and ECTV-Mos.

a<p>Genome sizes are from the first nt of the left CRS to the nt before the right CRS.</p>b<p>The ORF number includes the homolog of O3, which was not originally annotated in ECTV-Nav or ECTV-Mos.</p

Genome map of ERPV and comparison to ECTV-Naval.

<p>Left (LITR) and right (RITR) inverted terminal repeats are indicated by deep blue arrows. ORFs are in yellow and numbered from left to right with the direction of transcription indicated by the arrow. Direct repeats (DRs) are indicated in light blue. Single nucleotide polymorphisms (SNPs) are gold; insertions and deletions are indicated in red and purple, respectively, with single nt and larger changes by a thin oval and a diamond, respectively. Asterisks signify mutations that affect the predicted amino acid sequence.</p

Assembly of contigs and gap closure.

<p>(A) Five contigs were assembled de novo using 159,077 sequence reads generated by pyrosequencing, providing an estimated coverage of 60X with 5 gaps. (B) The gaps were filled by PCR and Sanger sequencing. Blue arrows indicate positions of primers used for PCR. Gaps 2 and 5 contained direct repeats (DRs) necessitating synthesis and sequencing of additional internal PCR fragments. DRI contains a 69 bp sequence repeated 2.3X; DRII contained an 85 bp sequence repeated 10.4X; and DRIII contained a 25 bp sequence repeated 7.0X. The non-repetitive I (NRI) and NRII sequences flank DRI. ORFs are indicated by numbered yellow arrows.</p

Representation of an orthopoxvirus genome.

<p>A typical genome consisting of a single dsDNA molecule with a concatemer resolution sequence (CRS), sets of direct repeats (DRI and DRII) and a hairpin loop on each inverted terminal repeat (ITR) is shown.</p

Unassigned ORFs with homology to CPXV proteins.

a<p>Similar in ECTV-Mos.</p>b<p>Similar in ECTV-Nav.</p>c<p>Missing from ECTV-Mos. Others are identical in the three genomes.</p><p>Abbreviation: aa, amino acids.</p

Contrasting antibody responses to intrasubtype superinfection with CRF02_AG

<div><p>HIV superinfection describes the sequential infection of an individual with two or more unrelated HIV strains. Intersubtype superinfection has been shown to cause a broader and more potent heterologous neutralizing antibody response when compared to singly infected controls, yet the effects of intrasubtype superinfection remain controversial. Longitudinal samples were analyzed phylogenetically for <i>pol</i> and <i>env</i> regions using Next-Generation Sequencing and envelope cloning. The impact of CRF02_AG intrasubtype superinfection was assessed for heterologous neutralization and antibody binding responses. We compared two cases of CRF02_AG intrasubtype superinfection that revealed complete replacement of the initial virus by superinfecting CRF02_AG variants with signs of recombination. NYU6564, who became superinfected at an early time point, exhibited greater changes in antibody binding profiles and generated a more potent neutralizing antibody response post-superinfection compared to NYU6501. In contrast, superinfection occurred at a later time point in NYU6501 with strains harboring significantly longer V1V2 regions with no observable changes in neutralization patterns. Here we show that CRF02_AG intrasubtype superinfection can induce a cross-subtype neutralizing antibody response, and our data suggest timing and/or superinfecting viral envelope characteristics as contributing factors. These results highlight differential outcomes in intrasubtype superinfection and provide the first insight into cases with CRF02_AG, the fourth most prevalent HIV-1 strain worldwide.</p></div

Timeline and clinical parameters of the two cases of intrasubtype CRF02_AG superinfection.

<p><b>A)</b> Plasma samples were collected from 2002 to 2014 for patients NYU6501 and NYU6564. Samples are shown in green along the timeline. Red indicates the time span when superinfection occurred. Blue indicates antiretroviral treatment (ART). <b>B)</b> Collection dates, viral load, CD4 cell counts, and the time post diagnosis for each sample used in the study (mths abbreviates for months when listed). Red shades indicate the first time point collected after superinfection occurred. Blue shades highlight samples taken when the patient was on ART. Time points after superinfection are in bold.</p

Heterologous neutralization responses in two cases of intrasubtype CRF02_AG superinfection.

<p><b>A</b>, <b>B)</b> Table of IC50 values that represent the plasma dilutions (<b>A</b>) or IgG concentrations (<b>B</b>) needed for 50% neutralization of the respective pseudovirus. IC50 values were calculated using nonlinear regression fits of the neutralization curves in GraphPad Prism and are illustrated in a color-coded scheme. Resistance to neutralization was assumed if the plasma or IgG sample could not reach 50% neutralization at the lowest plasma dilution (<1) or highest IgG concentration (>500 μg/mL), respectively, and is indicated in the table with a green shade. Breadth and potency values were calculated as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.ref010" target="_blank">10</a>]. Pseudoviruses are all tier 2 covering subtypes A, B, C, G, and CRF02_AG, with the exception of lab strain BaL.26, tier 1, subtype B. MLV was tested as negative control and to ensure absence of ART (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.s009" target="_blank">S9 Fig</a>)</b>. Time points filled in with pale red are after superinfection, pale blue after initiation of ART. IC50, breadth, and potency values are calculated using the averages of 2 or more experiments. <b>C)</b> Neutralization curves from plasma samples for NYU6501 & NYU6564 against pseudoviruses T250-4 and Q23.17, shown as the percent neutralization at the reciprocal plasma dilution. <b>D)</b> Neutralization curves from IgG samples for NYU6501 and NYU6564 against pseudoviruses T250-4, shown as the percent neutralization at the given IgG concentration.</p

Multiple amino acid alignments of V1V2 and V3 regions with NYU6501 and NYU6564 envelope consensus sequences pre and post superinfection.

<p>Alignments were made with consensus sequences generated from all functional Env clones per time point of NYU6501 and NYU6564 (according to phylogenetic analyses in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.g002" target="_blank">Fig 2</a></b>); two consensus sequences per time point were created when distinct populations were detected (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.g002" target="_blank">Fig 2</a></b>; see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.s002" target="_blank">S2 Fig</a></b>). Patient Env sequences were aligned with V1V2 and V3 antigens (≥10 fold change in EC50, <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.g004" target="_blank">Fig 4B</a></b>; <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.s014" target="_blank">S2 Table</a></b>) and reference strains of subtypes CRF02_AG, A, C and B, also used as pseudoviruses for neutralization. Red residues indicate a nonsynonymous substitution and blue residues indicate isofunctional mutations compared to the time point 1 consensus sequence before SI, thereby indicating changes post-SI. <b>A)</b> Patient V1V2 consensus sequences aligned with the V1V2 ZM109 antigen used for binding experiments and reference sequences. Green and yellow boxes indicate the residues that make up the glycan V2 region and the integrin binding site, respectively, located within the immunodominant V1V2 region. V1 and V2 loops are indicated with brackets. <b>B)</b> Patient V3 sequences compared with the V3 ZM109 antigen and reference sequences. The V3 crown residues are denoted underneath.</p