Characterization of a highly evolved vaccine-derived poliovirus type 3 isolated from sewage in Estonia

Abstract

Two types of vaccine-derived polioviruses have been recently designated to emphasize the different origins of the evolved viruses: circulating vaccine-derived polioviruses (cVDPV) associated with outbreaks of paralytic disease and strains isolated from chronically infected immunodeficient individuals (iVDPV). We describe here a type 3 VDPV (PV3/EST/02/E252; later E252) isolated from sewage collected in Tallinn, Estonia, in October 2002. Due to aberrant properties in subtyping, the virus was subjected to detailed characterization. Partial genomic sequencing suggested that the closest relative was the oral vaccine strain PV3/Sabin, but the two virus strains shared only 86.7% of the 900 nucleotides (nt) coding for the capsid protein VP1. Phylogenetic analysis of the nearly complete genome [nt 19 to poly(A)] revealed multiple nucleotide substitutions throughout the genome and a possible Sabin 3/Sabin 1-recombination junction site in the 2C coding region. A calculation based on the estimated mutation frequency of the P1 region of polioviruses suggested that the E252 virus might have replicated in one or more individuals for approximately 10 years. No persons chronically excreting poliovirus are known in Estonia. Amino acid substitutions were seen in all known antigenic sites, which was consistent with the observed aberrant antigenic properties of the virus demonstrated by both monoclonal antibodies and human sera from vaccinated children. In spite of the apparent transmission potential, no evidence was obtained for circulation of the virus in the Estonian population.

FIG. 1.

Similarity analysis of complete PV genomes. Analysis was done with a sliding window of 200 nt moving in steps of 20 nt, with all positions with a gap deleted, and the nucleotide similarity was plotted by using the JC model of nucleotide substitution. (a) E252 is compared against PV1/Sabin and PV3/Sabin (b) E252 is compared against 12 different poliovirus or HEV-C genomes. The GenBank accession numbers of the sequences used are as follows: PV3/Sabin, X00925; PV3/23127, X04468; PV3/Usol-d-bac, AJ293918; PV1/Sabin, V01150; PV1/DOR00013, AF405690; PV1/RUS1161-96, AF462419; PV2/Sabin, X00595; CV-A21, D00538; CV-A24, D90457; and PV1/66months, AJ132961.

FIG. 2.

Neighbor-joining tree of the nucleotide sequence relationships of the isolate E252 and other polioviruses and HEV-C in VP1 (a) and 3D polymerase (b) protein coding regions. The location of E252 is indicated by an arrow. Numbers at nodes represent reliability values for the internal branch (= bootstrap values). Values over 70% are shown. The scale bar indicates the evolutionary distance (0.01 = 1%). The GenBank accession numbers for the sequences are as follows: CV-A1, AF081293 and AF499635; CV-A11, AF081301 and AF499636; CV-A13, AF081303 and AF499637; CV-A15, AF081305 and AF499638; CV-A17, AF081306 and AF499639; CV-A18, AF081307 and AF499640; CV-A19, AF081308 and AF499641; CV-A20, AF081309 and AF499642; and CV-A22, AF081310 and AF499643. For information on other sequences, see the legend to Fig. 1.

FIG. 3.

Predicted molecular structure of the protomer E252. A space-fill model of a protomer (14) is shown at three different angles. The picture was generated with the RasWin Molecular Visualization Program (version 2.6) (36). (a) Front view. (b) Left-side view. (c) Right-side view. VP1, blue; VP2, yellow; VP3, red; VP4, green. A changed amino acid is shown by cyan, an antigenic site is in white, and a changed amino acid in an antigenic site is in violet. Identification of amino acids by the one-letter code: T, threonine; K, lysine. The first number after the one-letter code indicates the capsid protein: 1, VP1; 2, VP2; and 3, VP3. The three-number code indicates the position of the amino acid in a given capsid protein.

FIG. 4.

Serum neutralizing antibodies to PV3 strains E252 and Sabin in children vaccinated with IPV (A) or OPV (B). One serum sample from each child was analyzed for neutralizing antibodies by using a standard microneutralization assay with fourfold serum dilutions. The negative sera (titers of <4) were blotted, as they were 1:2.