Reproductive capacity of VDPV2 isolate 44624 and Sabin 2 strain at different temperatures (RCT marker).

<p>The RCT value is defined as the difference between the log 10 virus titer of the viral stock measured at the optimal temperature 36,5°C and supraoptimal temperature 40°C. The values are expressed as log 10 TCID50 / 0,1ml. Virus were considered thermosensitive if the ΔRCT value was greater or equal to 2, and thermo resistant when RCT value was inferior to 2.00.</p

Amino acid substitutions in the capsid protomer of isolate 44624.

<p>VP1, VP2, VP3 and VP4 are represented as a 3-dimensional structured protomer. The image was generated using the software Swiss-PdbViewer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152251#pone.0152251.ref024" target="_blank">24</a>], based on X-ray crystallographic analysis of type 2 poliovirus strain Lansing (Protein Data Bank accession number 1EAH.pdb) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152251#pone.0152251.ref023" target="_blank">23</a>]. Colour codes: Substitutions at known antigenic sites, brown. Substitutions elsewhere, pink. The BC-loop of VP1 is not visible in this model.</p

Molecular and Phenotypic Characterization of a Highly Evolved Type 2 Vaccine-Derived Poliovirus Isolated from Seawater in Brazil, 2014

<div><p>A type 2 vaccine-derived poliovirus (VDPV), differing from the Sabin 2 strain at 8.6% (78/903) of VP1 nucleotide positions, was isolated from seawater collected from a seaport in São Paulo State, Brazil. The P1/capsid region is related to the Sabin 2 strain, but sequences within the 5'-untranslated region and downstream of the P1 region were derived from recombination with other members of Human Enterovirus Species C (HEV-C). The two known attenuating mutations had reverted to wild-type (A481G in the 5'-UTR and Ile143Thr in VP1). The VDPV isolate had lost the temperature sensitive phenotype and had accumulated amino acid substitutions in neutralizing antigenic (NAg) sites 3a and 3b. The date of the initiating OPV dose, estimated from the number of synonymous substitutions in the capsid region, was approximately 8.5 years before seawater sampling, a finding consistent with a long time of virus replication and possible transmission among several individuals. Although no closely related type 2 VDPVs were detected in Brazil or elsewhere, this VDPV was found in an area with a mobile population, where conditions may favor both viral infection and spread. Environmental surveillance serves as an important tool for sensitive and early detection of circulating poliovirus in the final stages of global polio eradication.</p></div

Alignment of amino acids residues of neutralizing antigenic (NAg) sites for Sabin 2 (GenBank accession number AY184220) and isolate 44624.

<p>Amino acid positions are numbered according to Sabin 2 NAg1 (VP1 88–106), NAg2 (VP2 163–169; VP2 268–270; VP1 220–225), NAg3a (VP3 54–61; VP3 70–74; VP1 286–291) and NA3b (VP2 71–73; VP3 75–79).</p

Analysis of nucleotide differences between the genome of isolate 44624 and Sabin 2.

<p>Analysis of nucleotide differences between the genome of isolate 44624 and Sabin 2.</p

Schematic representation of the genome of recombinant aVDPV 44624, isolated in Brazil.

<p>Non-vaccine sequences (HEV-C species) are represented in light blue. Highly mutated Sabin 2 regions are represented in gray.</p

Recombination events predicted by RDP algorithms for the genome of isolate 44624 and putative parental sequences.

<p>Break points consist of the beginning nucleotide (BN) and the ending nucleotide (EN) of recombination fragment detected in the break point analysis. The figures are for isolate 44624. RDP software considers the major parent as the sequence closely related to that from which the greater part of the recombinant’s sequence may have been derived, and the minor parent is pointed as the sequence closely related to that from which sequences in the proposed recombinant region may have been derived.</p

One-step growth curve analysis of isolate 44624 in comparison with Sabin 2 in RD cells.

<p>Cells were infected at a MOI of 10 and incubated at 37 or 40°C. Total virus production at different times (0-12h) post-infection were determined by TCID 50 assays on RD cells. Each point represents the mean + standard deviation of virus titers from three different experiments.</p