Molecular and antigenic characterization of a highly evolved derivative of the type 2 oral poliovaccine strain isolated from sewage in Israel

Abstract

An unusual, highly diverged derivative of the Sabin type 2 oral poliovaccine (OPV) strain was recovered from environmental samples during routine screening for wild polioviruses. Virus was cultivated in L20B cells and then passaged on BGM cells at 40 degrees C (RCT [reproductive capacity at supraoptimal temperature]-positive marker) to select against most OPV strains. All but 1 of 25 RCT-positive OPV-derived environmental isolates were antigenically and genetically (>99.5% VP1 sequence match) similar to the respective Sabin strains. However, isolate PV2/4568-1/ISR98 (referred to below as 4568-1) escaped neutralization with Sabin 2-specific monoclonal antibodies and cross-adsorbed sera, and had multiple nucleotide substitutions (220 of 2,646; 8.3%) in the P1 capsid region. Fourteen of the 44 associated amino acid substitutions in the capsid mapped to neutralizing antigenic sites. Neutralizing titers in the sera of 50 Israeli children 15 years old were significantly lower to 4568-1 (geometric mean titer [GMT], 47) than to Sabin 2 (GMT, 162) or to the prototype wild strain, PV2/MEF-1/EGY42 (GMT, 108). Two key attenuating sites had also reverted in 4568-1 (A(481) to G in the 5' untranslated region and the VP1 amino acid I(143) to T), and the isolate was highly neurovirulent for transgenic mice expressing the poliovirus receptor (PVR-Tg21 mice). The extensive genetic divergence of 4568-1 from the parental Sabin 2 strain suggested that the virus had replicated in one or more people for approximately 6 years. The presence in the environment of a highly evolved, neurovirulent OPV-derived poliovirus in the absence of polio cases has important implications for strategies for the cessation of immunization with OPV following global polio eradication.

FIG. 1

Intratypic differentiation of type 2, RCT-positive poliovirus isolates by blot hybridization. Viral RNA was bound to nylon filters and hybridized with a DIG-labeled Sabin 2-specific VP1 probe (left blot) or a panenteroviral 5′-UTR probe (right blot). Hybrids were detected by chemiluminescence (6). Controls: A1, Sabin 1; A2, Sabin 2; A3, Sabin 3; B1, Mahoney; B2, PV2/MEF-1/EGY42; B3, PV3/Saukett/USA52. RCT-positive isolates: C1, PV2/4568-1/ISR98; C2, PV2/4586-5/PAL98; C3, PV2/4588-3/PAL98; D1, PV2/4623-1/ISR98; D2, PV2/4625-1/ISR98.

FIG. 2

Phylogenetic tree of type 2 RCT-positive sewage isolates. VP1 sequences of 4568-1 were compared with those of Sabin 2, five other Sabin 2-derived RCT-positive sewage isolates from Israel (including the type 2 antigenic variant VP2/4625-1/ISR98), two isolates from immunodeficient VAPP patients in the United States (PV2/0715/USA91 and PV2/3739A/USA92), a divergent type 2 isolate from a patient in Peru (PV2/7834/PER83), two recent isolates from children with acute flaccid paralysis in Brazil (PV2/7089/BRA96 and PV2/7717/BRA97), the reference wild type 2 IPV strain, PV2/MEF-1/EGY42, and a wild type 2 isolate from a 1998 polio case in India (PV2/7570/IND98).

FIG. 3

Amino acid substitutions in NAg sites of 4568-1. Nonhomologous amino acids are indicated below the consensus sequence for each NAg site. Amino acid positions are numbered according to that for Sabin 2 (26).