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

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.     

2010年山东省环境污水中脊髓灰质炎病毒的监测及其基因特征分析

本研究在山东省开展了脊髓灰质炎病毒(Poliovirus,PV)的外环境监测,从济南、临沂两地采集污水标本,浓缩处理后进行病毒分离,对分离到的PV采用中和试验进行血清定型,并对其VP1及3D区进行序列测定,分析其基因突变和重组情况。2010年,共采集污水标本32份,PV阳性10份,阳性率31.3%;分离到18株PV(PV1型3株,PV2型9株,PV3型6株),均为疫苗相关株,VP1完整编码区核苷酸变异数在0~4个之间,在3株PV2型病毒和4株PV3型病毒的基因组中发现重组;对VP1区影响神经毒力的减毒位点分析发现,PV1型病毒中有1株在nt 2 749发生突变(A→G),PV2型病毒中有1株在nt2 908发生A→G突变,3株在nt2 909发生U→C突变,6株PV3型病毒全部在nt2 493发生C→U突变。环境污水中可以分离到PV,其基因重组率和主要减毒位点的回复突变率较高,未发现脊灰野毒株和疫苗衍生株脊灰病毒(Vaccine-derived poliovirus,VDPV)。     

Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing.

Intracerebral inoculation of mice with poliovirus type 2 Lansing induces a fatal paralysis, while most other poliovirus strains are unable to cause disease in the mouse. To determine the molecular basis for Lansing virus neurovirulence, we determined the complete nucleotide sequence of the Lansing viral genome from cloned cDNA. The deduced amino acid sequence was compared with that of two mouse-avirulent strains. There are 83 amino acid differences between the Lansing and Sabin type 2 strain and 179 differences between the Lansing and Mahoney type 1 strain scattered throughout the genome. To further localize Lansing sequences important for mouse neurovirulence, four intertypic recombinants were isolated by exchanging DNA restriction fragments between the Lansing 2 and Mahoney 1 infectious poliovirus cDNA clones. Plasmids were transfected into HeLa cells, and infectious recombinant viruses were recovered. All four recombinant viruses, which contained the Lansing capsid region and different amounts of the Mahoney genome, were neurovirulent for 18- to 21-day-old Swiss-Webster mice by the intracerebral route. The genome of neurovirulent recombinant PRV5.1 contained only nucleotides 631 to 3413 from Lansing, encoding primarily the viral capsid proteins. Therefore, the ability of Lansing virus to cause paralysis in mice is due to the viral capsid. The Lansing capsid sequence differs from that of the mouse avirulent Sabin 2 strain at 32 of 879 amino acid positions: 1 in VP4, 5 in VP2, 4 in VP3, and 22 in VP1.     

Mapping of attenuating sequences of an avirulent poliovirus type 2 strain.

A mouse model for poliomyelitis was used to identify genomic sequences that attenuate neurovirulence of poliovirus strain P2/P712. This type 2 strain is avirulent in primates and mice yet grows as well as virulent strains in cell culture. The approach used was to exchange portions of the genome of the mouse-virulent P2/Lansing strain with the corresponding region from P2/P712 to identify sequences that could attenuate Lansing neurovirulence in mice. A full-length infectious cDNA of P2/P712 was assembled and used to construct recombinants between P2/P712 and P2/Lansing. The results of neurovirulence testing of 11 recombinants indicated that strong attenuating determinants are located in the 5' noncoding region of P2/P712 and a region encoding capsid protein VP1 and 2Apro, 2B, and part of 2C. An attenuating determinant was further localized to between nucleotides 456 and 628 of P2/P712. A third sequence from P2/P712, nucleotides 752 to 2268, encoding VP4, VP2, and part of VP3, was weakly attenuating. The sequence from nucleotide 4454, approximately halfway through the 2C-coding region, to the end of the P2/P712 genome did not contain attenuating determinants. Nucleotide sequence analysis revealed that P2/P712 differs from the type 2 Sabin vaccine strain by only 22 nucleotides. Six differences lead to amino acid changes in the coding region, and four differences are in the 5' noncoding region. These studies show that, like the type 1 and type 3 Sabin vaccine strains, the attenuated type 2 strain P712 contains multiple attenuating sequences, including strongly attenuating sequences in the 5' noncoding region of the genome.     

Serial recombination during circulation of type 1 wild-vaccine recombinant polioviruses in China

Type 1 wild-vaccine recombinant polioviruses sharing a 367-nucleotide (nt) block of Sabin 1-derived sequence spanning the VP1 and 2A genes circulated widely in China from 1991 to 1993. We surveyed the sequence relationships among 34 wild-vaccine recombinants by comparing six genomic intervals: the conserved 5'-untranslated region (5'-UTR) (nt 186 to 639), the hypervariable portion of the 5'-UTR (nt 640 to 742), the VP4 and partial VP2 genes (nt 743 to 1176), the VP1 gene (nt 2480 to 3385), the 2A gene (nt 3386 to 3832), and the partial 3D gene (nt 6011 to 6544). The 5'-UTR, capsid (VP4-VP2 and VP1), and 2A sequence intervals had similar phylogenies. By contrast, the partial 3D sequences could be distributed into five divergent genetic classes. Most (25 of 34) of the wild-vaccine recombinant isolates showed no evidence of additional recombination beyond the initial wild-Sabin recombination event. Eight isolates from 1992 to 1993, however, appear to be derived from three independent additional recombination events, and one 1993 isolate was derived from two consecutive events. Complete genomic sequences of a representative isolate for each 3D sequence class demonstrated that these exchanges had occurred in the 2B, 2C, and 3D genes. The 3D gene sequences were not closely related to those of the Sabin strains or 53 diverse contemporary wild poliovirus isolates from China, but all were related to the 3D genes of species C enteroviruses. The appearance within approximately 2.5 years of five recombinant classes derived from a single ancestral infection illustrates the rapid emergence of new recombinants among circulating wild polioviruses.     

An Outbreak of Type Π Vaccine-Derived Poliovirus in Sichuan Province,China: Emergence and Circulation in an Under-Immunized Population

Background

During August 2011–February 2012, an outbreak of type Π circulating vaccine-derived poliovirus (cVDPVs) occurred in Sichuan Province, China.

Methods

A field investigation of the outbreak was conducted to characterize outbreak isolates and to guide emergency response. Sequence analysis of poliovirus capsid protein VP1 was performed to determine the viral propagation, and a coverage survey was carried out for risk assessment.

Results

One clinical compatible polio case and three VDPV cases were determined in Ngawa County, Ngawa Tibetan and Qiang Autonomous Prefecture, Sichuan Province. Case patients were unimmunized children, 0.8–1 years old. Genetic sequencing showed that the isolates diverged from the VP1 region of the type Π Sabin strain by 5–12 nucleotides (nt) and shared the same 5 nt VP1 substitutions, which indicate single lineage of cVDPVs. Of the 7 acute flaccid paralysis cases (all>6 months) reported in Ngawa Prefecture in 2011, 4 (57.1%) cases (including 2 polio cases) did not receive oral attenuated poliovirus vaccine. Supplementary immunization activities (SIAs) were conducted in February–May, 2012, and the strain has not been isolated since.

Conclusion

High coverage of routine immunization should be maintained among children until WPV transmission is globally eradicated. Risk assessments should be conducted regularly to pinpoint high risk areas or subpopulations, with SIAs developed if necessary.     

Isolation of an intertypic poliovirus capsid recombinant from a child with vaccine-associated paralytic poliomyelitis

The isolation of a capsid intertypic poliovirus recombinant from a child with vaccine-associated paralytic poliomyelitis is described. Virus 31043 had a Sabin-derived type 3-type 2-type 1 recombinant genome with a 5'-end crossover point within the capsid coding region. The result was a poliovirus chimera containing the entire coding sequence for antigenic site 3a derived from the Sabin type 2 strain. The recombinant virus showed altered antigenic properties but did not acquire type 2 antigenic characteristics. The significance of the presence in nature of such poliovirus chimeras and the consequences for the current efforts to detect potentially dangerous vaccine-derived poliovirus strains are discussed in the context of the global polio eradication initiative.     

Complete genomic characterization of an intertypic Sabin 3/Sabin 2 capsid recombinant

The genetic properties of strain K/2002, isolated from fecal samples of a 7-month-old child who had received his first oral poliovirus vaccine (OPV) dose at the age of 3 months, are described. Preliminary sequencing characterization of isolate K/2002 revealed an S3/S2 recombination event at the 3' end of the VP1 coding region. A recombination event resulted in the introduction of six Sabin 2 amino acid residues in a Sabin 3 genomic background. Furthermore, mutations associated with loss of the attenuated phenotype of Sabin 3 strains have been identified in the genome of isolate K/2002. The data presented here emphasize the need for careful planning of vaccination strategies, which involve stopping OPV administration in regions that are certified to be polio-free.     

Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine.

To identify determinants of attenuation in the poliovirus type 1 Sabin vaccine strain, a series of recombinant viruses were constructed by using infectious cDNA clones of the virulent type 1 poliovirus P1/Mahoney and the attenuated type 1 vaccine strain P1/Sabin. Intracerebral inoculation of these viruses into transgenic mice which express the human receptor for poliovirus identified regions of the genome that conferred reduced neurovirulence. Exchange of smaller restriction fragments and site-directed mutagenesis were used to identify the nucleotide changes responsible for attenuation. P1/Sabin mutations at nucleotides 935 of VP4, 2438 of VP3, and 2795 and 2879 of VP1 were all shown to be determinants of attenuation. The recombinant viruses and site-directed mutants were also used to identify the nucleotide changes which are involved in the temperature sensitivity of P1/Sabin. Determinants of this phenotype in HeLa cells were mapped to changes at nucleotides 935 of VP4, 2438 of VP3, and 2741 of VP1. The 3Dpol gene of P1/Sabin, which contains three amino acid differences from its parent P1/Mahoney, also contributes to the temperature sensitivity of P1/Sabin; however, mutants containing individual amino acid changes grew as well as P1/Mahoney at elevated temperatures, suggesting that either some combination or all three changes are required for temperature sensitivity. In addition, the 3'-noncoding region of P1/Sabin augments the temperature-sensitive phenotype conferred by 3Dpol. Although nucleotide 2741, 3Dpol, and the 3'-noncoding region of P1/Sabin contribute to the temperature sensitivity of P1/Sabin, they do not contribute to attenuation in transgenic mice expressing the poliovirus receptor, demonstrating that determinants of attenuation and temperature sensitivity can be genetically separated.     

Nucleotide sequence of a neurovirulent variant of the type 2 oral poliovirus vaccine.

Infectious cDNAs of the Sabin type 2 poliovirus vaccine virus and a vaccine-derived neurovirulent type 2 strain (P2/117) have been cloned in Escherichia coli. Nucleotide sequence analysis revealed that P2/117 differs from the vaccine strain by just 23 point mutations. Three occur in the 5' noncoding region. The remainder result in a total of 5 coding changes located in VP1, VP4, 2B, and 3D. The likely role of these mutations in the evolution to neurovirulence is discussed.     

Circulation of endemic type 2 vaccine-derived poliovirus in Egypt from 1983 to 1993

From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5' untranslated region (5' UTR) and noncapsid- 3' UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide.     

Retrospective Characterization of a Vaccine-Derived Poliovirus Type 1 Isolate from Sewage in Greece

Retrospective molecular and phenotypic characterization of a vaccine-derived poliovirus (VDPV) type 1 isolate (7/b/97) isolated from sewage in Athens, Greece, in 1997 is reported. VP1 sequencing of this isolate revealed 1.87% divergence from the VP1 region of reference strain Sabin 1, while further genomic characterization of isolate 7/b/97 revealed a recombination event in the nonstructural part of the genome between a vaccine strain and a nonvaccine strain probably belonging to Enterovirus species C. Amino acid substitutions commonly found in previous studies were identified in the capsid coding region of the isolate, while most of the attenuation and temperature sensitivity determinants were reverted. The ultimate source of isolate 7/b/97 is unknown. The recovery of such a highly divergent derivative of a vaccine strain emphasizes the need for urgent implementation of environmental surveillance as a supportive procedure in the polio surveillance system even in countries with high rates of OPV coverage in order to prevent cases or even outbreaks of poliomyelitis that otherwise would be inevitable.     

Molecular evolution of a type 1 wild-vaccine poliovirus recombinant during widespread circulation in China

Type 1 wild-vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. We compared the sequences of 34 recombinant isolates over the 1,353-nucleotide (nt) genomic interval (nt 2480 to 3832) encoding the major capsid protein, VP1, and the protease, 2A. All recombinants had a 367-nt block of sequence (nt 3271 to 3637) derived from the Sabin 1 oral poliovirus vaccine strain spanning the 3'-terminal sequences of VP1 (115 nt) and the 5' half of 2A (252 nt). The remaining VP1 sequences were closely (up to 99.5%) related to those of a major genotype of wild type 1 poliovirus endemic to China up to 1994. In contrast, the non-vaccine-derived sequences at the 3' half of 2A were more distantly related (<90% nucleotide sequence match) to those of other contemporary wild polioviruses from China. The vaccine-derived sequences of the earliest (April 1991) isolates completely matched those of Sabin 1. Later isolates diverged from the early isolates primarily by accumulation of synonymous base substitutions (at a rate of approximately 3.7 x 10(-2) substitutions per synonymous site per year) over the entire VP1-2A interval. Distinct evolutionary lineages were found in different Chinese provinces. From the combined epidemiologic and evolutionary analyses, we propose that the recombinant virus arose during mixed infection of a single individual in northern China in early 1991 and that its progeny spread by multiple independent chains of transmission into some of the most populous areas of China within a year of the initiating infection.     

Evolution of Type 2 Vaccine Derived Poliovirus Lineages. Evidence for Codon-Specific Positive Selection at Three Distinct Locations on Capsid Wall

Partial sequences of 110 type 2 poliovirus strains isolated from sewage in Slovakia in 2003–2005, and most probably originating from a single dose of oral poliovirus vaccine, were subjected to a detailed genetic analysis. Evolutionary patterns of these vaccine derived poliovirus strains (SVK-aVDPV2) were compared to those of type 1 and type 3 wild poliovirus (WPV) lineages considered to have a single seed strain origin, respectively. The 102 unique SVK-aVDPV VP1 sequences were monophyletic differing from that of the most likely parental poliovirus type 2/Sabin (PV2 Sabin) by 12.5–15.6%. Judging from this difference and from the rate of accumulation of synonymous transversions during the 22 month observation period, the relevant oral poliovirus vaccine dose had been administered to an unknown recipient more than 12 years earlier. The patterns of nucleotide substitution during the observation period differed from those found in the studied lineages of WPV1 or 3, including a lower transition/transversion (Ts/Tv) bias and strikingly lower Ts/Tv rate ratios at the 2^nd codon position for both purines and pyrimidines. A relatively low preference of transitions at the 2^nd codon position was also found in the large set of VP1 sequences of Nigerian circulating (c)VDPV2, as well as in the smaller sets from the Hispaniola cVDPV1 and Egypt cVDPV2 outbreaks, and among aVDPV1and aVDPV2 strains recently isolated from sewage in Finland. Codon-wise analysis of synonymous versus non-synonymous substitution rates in the VP1 sequences suggested that in five codons, those coding for amino acids at sites 24, 144, 147, 221 and 222, there may have been positive selection during the observation period. We conclude that pattern of poliovirus VP1 evolution in prolonged infection may differ from that found in WPV epidemics. Further studies on sufficiently large independent datasets are needed to confirm this suggestion and to reveal its potential significance.     

Retrospective characterization of a vaccine-derived poliovirus type 1 isolate from sewage in Greece

Retrospective molecular and phenotypic characterization of a vaccine-derived poliovirus (VDPV) type 1 isolate (7/b/97) isolated from sewage in Athens, Greece, in 1997 is reported. VP1 sequencing of this isolate revealed 1.87% divergence from the VP1 region of reference strain Sabin 1, while further genomic characterization of isolate 7/b/97 revealed a recombination event in the nonstructural part of the genome between a vaccine strain and a nonvaccine strain probably belonging to Enterovirus species C. Amino acid substitutions commonly found in previous studies were identified in the capsid coding region of the isolate, while most of the attenuation and temperature sensitivity determinants were reverted. The ultimate source of isolate 7/b/97 is unknown. The recovery of such a highly divergent derivative of a vaccine strain emphasizes the need for urgent implementation of environmental surveillance as a supportive procedure in the polio surveillance system even in countries with high rates of OPV coverage in order to prevent cases or even outbreaks of poliomyelitis that otherwise would be inevitable.     

Expression of poliovirus complementary DNA coding for viral antigenic determinants in Escherichia coli

DNA sequences coding for the immunogenic capsid protein VP1 and/or VP3 of poliovirus strain LSc-2ab (Sabin 1) were prepared by digesting the cloned complementary DNA with restriction endonuclease PstI. The DNA fragments were inserted into the unique PstI site of Escherichia coli plasmid vectors pBR322, pKT 280 and/or pKT 287 that lay in the region expressed under control of the penicillinase promoter system. In the expression vectors, poliovirus sequences were designed to be read in phase and therefore to be expressed as fusion proteins with the bacterial peptides. In addition, the Escherichia coli tryptophan operon promoter-operator system was inserted upstream of the penicillinase system to obtain stronger expression of the poliovirus sequences. Escherichia coli transformed with these plasmids appeared to produce the antigenic polypeptides, which were detected by immunoprecipitation with antibodies to capsid proteins VP1 and/or VP3 followed by SDS-polyacrylamide gel electrophoresis.     

Identification of a consistent pattern of mutations in neurovirulent variants derived from the sabin vaccine strain of poliovirus type 2.

Complete nucleotide sequencing of the RNAs of two unrelated neurovirulent isolates of Sabin-related poliovirus type 2 revealed that two nucleotides and one amino acid (amino acid 143 in the major capsid protein VP1) consistently departed from the sequences of the nonneurovirulent poliovirus type 2 712 and Sabin vaccine strains. This pattern of mutation appeared to be a feature common to all neurovirulent variants of poliovirus type 2.     

In vitro phenotypic markers of a poliovirus recombinant constructed from infectious cDNA clones of the neurovirulent Mahoney strain and the attenuated Sabin 1 strain.

Infectious cDNA corresponding to the entire genome of the attenuated Sabin strain of type 1 poliovirus has been inserted into EcoRI site of bacterial plasmid pBR325. Two consecutive PstI fragments (nucleotide positions 1814 to 3421) of the infectious cDNA of the Sabin 1 strain were replaced by the corresponding DNA fragments prepared from an infectious DNA clone of the genome of the virulent Mahoney strain of poliovirus type 1. The exchanged segment encodes capsid protein VP1 and part of capsid protein VP3, a region in which a large number of amino acid differences between the attenuated Sabin and the parental, neurovirulent Mahoney strain cluster. The recombinant virus was obtained by DNA transfection of HeLa S3 cells, and several in vitro phenotypes of the virus were compared with those of the parental viruses. The recombinant virus was recognized by a neutralizing monoclonal antibody specific to the Mahoney strain. Growth of the Sabin strain of poliovirus has been shown to be quite dependent upon the bicarbonate concentration (d marker). The growth of the recombinant virus, however, was not highly dependent upon the concentration of bicarbonate in cell culture media, and thus resembled that of the Mahoney strain. On the other hand, the temperature-sensitive multiplication (rct marker) and the small-plaque morphology of the recombinant virus corresponded to the phenotype of the Sabin 1 strain. The in vitro recombination of infectious cDNA clones of genomic RNA and subsequent analysis of the growth properties of the recombinant virus have allowed us to correlate specific mutations in the genome of an RNA virus with certain biological characteristics of that virus.     

Natural recombination event within the capsid genomic region leading to a chimeric strain of human enterovirus B

Recombination between two strains is a known phenomenon for enteroviruses replicating within a single cell. We describe a recombinant strain recovered from human stools, typed as coxsackievirus B4 (CV-B4) and CV-B3 after partial sequencing of the VP1 and VP2 coding regions, respectively. The strain was neutralized by a polyclonal CV-B3-specific antiserum but not by a CV-B4-specific antiserum. The nucleotide sequence analysis of the whole structural genomic region showed the occurrence of a recombination event at position 1950 within the VP3 capsid gene, in a region coding for the 2b antigenic site previously described for CV-B3. This observation evidences for the first time the occurrence of an interserotypic recombination within the VP2-VP3-VP1 capsid region between two nonpoliovirus enterovirus strains. The neutralization pattern suggests that the major antigenic site is located within the VP2 protein.     

Antiviral activity of 3(2H)- and 6-chloro-3(2H)-isoflavenes against highly diverged, neurovirulent vaccine-derived, type2 poliovirus sewage isolates

Background

Substituted flavanoids interfere with uncoating of Enteroviruses including Sabin-2 polio vaccine strains. However flavanoid resistant and dependent, type-2 polio vaccine strains (minimally-diverged), emerged during in vitro infections. Between 1998–2009, highly-diverged (8 to >15%) type-2, aVDPV₂s, from two unrelated persistent infections were periodically isolated from Israeli sewage.

Aim

To determine whether highly evolved aVDPV₂s derived from persistent infections retained sensitivity to isoflavenes.

Methods

Sabin-2 and ten aVDPV₂ isolates from two independent Israeli sources were titered on HEp2C cells in the presence and absence of 3(2H)- Isoflavene and 6-chloro-3(2H)-Isoflavene. Neurovirulence of nine aVDPV₂s was measured in PVR-Tg-21 transgenic mice. Differences were related to unique amino acid substitutions within capsid proteins.

Principal Findings

The presence of either flavanoid inhibited viral titers of Sabin-2 and nine of ten aVDPV₂s by one to two log₁₀. The tenth aVDPV₂, which had unique amino acid substitution distant from the isoflavene-binding pocket but clustered at the three- and five-fold axies of symmetry between capsomeres, was unaffected by both flavanoids. Genotypic neurovirulence attenuation sites in the 5′UTR and VP1 reverted in all aVDPV₂s and all reacquired a full neurovirulent phenotype except one with amino acid substitutions flanking the VP1 site.

Conclusion

Both isoflavenes worked equally well against Sabin 2 and most of the highly-diverged, Israeli, aVDPV₂s isolates. Thus, functionality of the hydrophobic pocket may be unaffected by selective pressures exerted during persistent poliovirus infections. Amino acid substitutions at sites remote from the drug-binding pocket and adjacent to a neurovirulence attenuation site may influence flavanoid antiviral activity, and neurovirulence, respectively.