The 2010 outbreak of poliomyelitis in Tajikistan: epidemiology and lessons learnt.

A large outbreak of poliomyelitis, with 463 laboratory-confirmed and 47 polio-compatible cases, took place in 2010 in Tajikistan. Phylogenetic analysis of the viral VP1 gene suggested a single importation of wild poliovirus type 1 from India in late 2009, its further circulation in Tajikistan and expansion into neighbouring countries, namely Kazakhstan, Russia, Turkmenistan and Uzbekistan. Whole-genome sequencing of 14 isolates revealed recombination events with enterovirus C with cross-overs within the P2 region. Viruses with one class of recombinant genomes co-circulated with the parental virus, and representatives of both caused paralytic poliomyelitis. Serological analysis of 327 sera from acute flaccid paralysis cases as well as from patients with other diagnoses and from healthy people demonstrated inadequate immunity against polio in the years preceding the outbreak. Evidence was obtained suggesting that vaccination against poliomyelitis, in rare cases, may not prevent the disease. Factors contributing to the peculiarities of this outbreak are discussed. The outbreak emphasises the necessity of continued vaccination against polio and the need, at least in risk areas, of quality control of this vaccination through well planned serological surveillance.

Neurovirulence of Sabin 1-derived polioviruses isolated from an immunodeficient patient with prolonged viral excretion.

We have analysed the neurovirulence of Sabin 1-derived isolates which persisted more than nine years in an immunodeficient patient in the U.S.A. Samples were collected from stool specimens at days 11 (St1), 23 (St2), 48 (St3), 126 (St5), and 200 (St7) after the onset of paralysis. Critical nucleotides associated with the reversion of virulence were examined. All the isolates had the substitutions at nucleotide positions 480 (G to A) in the 5'-non-coding region (NCR), 2438 (A to U) in VP3, 2795 (A to G) in VP1, and 6203 (C to U) in 3D. Serially diluted samples were injected intracerebrally to transgenic mice harbouring the human poliovirus receptor gene. Samples St2, 3, 5 and 7 showed typical virulent characters in transgenic mice, whereas the sample ST1 showed intermediate neurovirulence. It seemed that there were two variant viruses providing for a major (M) and a minor (m) populations. After disappearance of the m-variant, samples obtained at the later stages showed neurovirulence almost equivalent to that of the Mahoney strain. Thus, the Sabin 1 strain evolved towards full neuropathogenicity after long-term replication in humans by accumulating mutations. Therefore, OPV-vaccination of immunodeficient persons should be avoided.

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.

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

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.

Intratypic differentiation & partial nucleotide sequencing of poliovirus isolates of northern India.

The potential resolving power of molecular epidemiological studies has enhanced the precision and reliability of poliovirus (PV) surveillance. PV has an error prone RNA polymerase responsible for rapid evolution of genome (approximately 10(-2) nt substitution/site/year), during inter and intra-human passages. The present study included a serotyped panel of 60 PV (42 PV type-1, 13 PV type-2 and 5 PV type-3) isolated during 1997. They were differentiated into vaccine (Sabin) and wild strains by two methods viz., genotype specific RNA probe hybridization (Rpro-Hy) based on genotypic variability; and ELISA that uses cross-absorbed antiserum (Pab-E) based on phenotypic variability. For obtaining information on molecular epidemiology, partial nucleotide sequencing (VP1/2A region) of five clinical PV isolates was also done. Three of the 60 isolates (two PV type-1 and one PV type-3) intratyped, could not be differentiated correctly by either method. Genotypic characterization of PV isolates was done for confirmation of intratyping results. All five wild PV1 sequenced belonged to the same genotype (> 85% homology) and sequence divergence among the strains was < or = 4.5 per cent. This indicated circulation of a single genetic lineage in the area.

Evolution of circulating wild poliovirus and of vaccine-derived poliovirus in an immunodeficient patient: a unifying model.

We determined nucleotide sequences of the VP1 and 2AB genes and portions of the 2C and 3D genes of two evolving poliovirus lineages: circulating wild viruses of T geotype and Sabin vaccine-derived isolates from an immunodeficient patient. Different regions of the viral RNA were found to evolve nonsynchronously, and the rate of evolution of the 2AB region in the vaccine-derived population was not constant throughout its history. Synonymous replacements occurred not completely randomly, suggesting the need for conservation of certain rare codons (possibly to control translation elongation) and the existence of unidentified constraints in the viral RNA structure. Nevertheless the major contribution to the evolution of the two lineages came from linear accumulation of synonymous substitutions. Therefore, in agreement with current theories of viral evolution, we suggest that the majority of the mutations in both lineages were fixed as a result of successive sampling, from the heterogeneous populations, of random portions containing predominantly neutral and possibly adverse mutations. As a result of such a mode of evolution, the virus fitness may be maintained at a more or less constant level or may decrease unless more-fit variants are stochastically generated. The proposed unifying model of natural poliovirus evolution has important implications for the epidemiology of poliomyelitis.

Resolution of the pathways of poliovirus type 1 transmission during an outbreak.

An outbreak of poliomyelitis with 20 cases occurred in Israel, Gaza, and the West Bank from October 1987 to October 1988. The wild type 1 poliovirus associated with the outbreak was most closely related to viruses found in the Nile Delta. The epidemiologic links among patients involved in the outbreak and patients with community-acquired infections during the outbreak were inferred from the evolutionary relationships among isolates of the outbreak virus. Complete VP1 sequences (906 nucleotides) were determined for 12 clinical and 4 sewage isolates. A total of 58 nucleotide differences were found among the 16 isolates; 74% of all substitutions were synonymous third-position transitions. An evolutionary tree, representing both the pathways of VP1 sequence evolution and the inferred chains of virus transmission during the outbreak, was constructed under the assumption that each substitution had occurred only once. The combined epidemiologic and molecular data suggest that a single founder strain was introduced into Israel from the vicinity of Gaza in the fall of 1987. Poliovirus circulation was apparently localized to southern communities during the winter and spread north by the following summer into the Hadera subdistrict of Israel, where it radiated via multiple chains of transmission into other communities in northern Israel and the West Bank. The close sequence matches (>99%) between clinical and sewage isolates from the same communities confirm the utility of environmental sampling as a tool for monitoring wild poliovirus circulation.

Detection of poliovirus circulation by environmental surveillance in the absence of clinical cases in Israel and the Palestinian authority.

The global eradication of poliomyelitis, believed to be achievable around the year 2000, relies on strategies which include high routine immunization coverage and mass vaccination campaigns, along with continuous monitoring of wild-type virus circulation by using the laboratory-based acute flaccid paralysis (AFP) surveillance. Israel and the Palestinian Authority are located in a geographical region in which poliovirus is still endemic but have been free of poliomyelitis since 1988 as a result of intensive immunization programs and mass vaccination campaigns. To monitor the wild-type virus circulation, environmental surveillance of sewage samples collected monthly from 25 to 30 sites across the country was implemented in 1989 and AFP surveillance began in 1994. The sewage samples were processed in the laboratory with a double-selective tissue culture system, which enabled economical processing of large number of samples. Between 1989 and 1997, 2,294 samples were processed, and wild-type poliovirus was isolated from 17 of them in four clusters, termed "silent outbreaks," in September 1990 (type 3), between May and September 1991 (type 1), between October 1994 and June 1995 (type 1), and in December 1996 (type 1). Fifteen of the 17 positive samples were collected in the Gaza Strip, 1 was collected in the West Bank, and 1 was collected in the Israeli city of Ashdod, located close to the Gaza Strip. The AFP surveillance system failed to detect the circulating wild-type viruses. These findings further emphasize the important role that environmental surveillance can play in monitoring the eradication of polioviruses.

Prolonged replication of a type 1 vaccine-derived poliovirus in an immunodeficient patient.

VP1 sequences were determined for poliovirus type 1 isolates obtained over a 189-day period from a poliomyelitis patient with common variable immunodeficiency syndrome (a defect in antibody formation). The isolate from the first sample, taken 11 days after onset of paralysis, contained two poliovirus populations, differing from the Sabin 1 vaccine strain by approximately 10%, differing from diverse type 1 wild polioviruses by 19 to 24%, and differing from each other by 5.5% of nucleotides. Specimens taken after day 11 appeared to contain only one major poliovirus population. Evolution of VP1 sequences at synonymous third-codon positions occurred at an overall rate of approximately 3.4% per year over the 189-day period. Assuming this rate to be constant throughout the period of infection, the infection was calculated to have started approximately 9.3 years earlier. This estimate is about the time (6. 9 years earlier) the patient received his last oral poliovirus vaccine dose, approximately 2 years before the diagnosis of immunodeficiency. These findings may have important implications for the strategy to eliminate poliovirus immunization after global polio eradication.

Population biology, evolution, and immunology of vaccination and vaccination programs.

The purpose of prophylactic vaccination is to reduce morbidity and mortality in a population. Many questions related to the design of vaccines and vaccination programs require a population standpoint for their sharp formulation and laboratory and field studies to understand their immunologic background. Practical suggestions of the workshop included increased studies of age-specific immunity, better immunoepidemiologic surveillance, better design of efficacy studies, and more systematic sampling of parasite strains to study the evolutionary pressure exerted by vaccines. Theoretical immunology has much to contribute. One of the realizations of the workshop was the value of a strong interdisciplinary approach in vaccine development, utilizing relevant contributions from immunology, population biology, mathematical modeling, epidemiology, molecular biology, and virology.

Serotype-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residues at positions of codon degeneracy.

We have developed a method for determining the serotypes of poliovirus isolates by PCR. Three sets of serotype-specific antisense PCR-initiating primers (primers seroPV1A, seroPV2A, and seroPV3A) were designed to pair with codons of VP1 amino acid sequences that are conserved within but that differ across serotypes. The sense polarity primers (primers seroPV1S, seroPV2S, and seroPV3S) matched codons of more conserved capsid sequences. The primers contain mixed-base and deoxyinosine residues to compensate for the high rate of degeneracy of the targeted codons. The serotypes of all polioviruses tested (48 vaccine-related isolates and 110 diverse wild isolates) were correctly identified by PCR with the serotype-specific primers. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions with any of the three primer sets. These primers are useful for the rapid screening of poliovirus isolates and for determining the compositions of cultures containing mixtures of poliovirus serotypes.

Genotype-specific RNA probes for direct identification of wild polioviruses by blot hybridization.

We have developed RNA probes for the direct identification of wild poliovirus isolates by blot hybridization. The probes are complementary to sequences of the first 30 to 32 codons of VP1, which evolve more extensively (approximately 1.5-fold) than the rest of VP1. To illustrate our general approach, we describe the design of probes specific to each of four major genotypes recently endemic (1981 to 1991) to the Americas: Andean type 1, Brazil type 1, Brazil type 3, and Central America-Mexico type 3. A wild isolate of each genotype was selected according to molecular and epidemiologic criteria to be representative of the principal lineages in circulation. Variable VP1 sequences of the representative isolates were amplified by the reverse transcriptase PCR and were inserted into a plasmid vector containing a T7 promoter. The in vitro transcripts, labeled with digoxigenin, served as probes. These formed stable hybrids only with RNAs of isolates of the corresponding genotypes. Hybrids were detected by a sensitive chemiluminescence assay, capable under normal diagnostic conditions of detecting specific wild poliovirus sequences in samples containing up to a 100-fold excess of Sabin vaccine strain-related sequences of the same serotype.

Poliomyelitis in Angola: current status and implications for poliovirus eradication in Southern Africa.

As part of emergency assistance to the Ministry of Health (MOH), national surveillance data for poliomyelitis and charts of cases at the national rehabilitation hospital were reviewed. Poliomyelitis patients admitted to Angola's main pediatric hospital were examined. A mean of 86 cases of poliomyelitis/year were reported in Angola during 1989-1994. Review of records from non-MOH sources uncovered another 74 cases, primarily from areas outside governmental control. Hospital chart reviews revealed that 80% of the cases were children <3 years of age, mainly unvaccinated. Molecular analyses of isolates from cases in Luanda and at the Angola-Namibia border suggest that these isolates are closely related and that > or = 2 strains of wild poliovirus type 1 are circulating currently in Angola. This investigation confirms that poliomyelitis has remained endemic in Angola since independence in 1975. It affects primarily young and unvaccinated children. Control of poliomyelitis in Angola is essential to expand the polio-free zone in southern Africa.

Eradication of wild poliovirus from the Americas: wild poliovirus surveillance--laboratory issues.

The Pan American Regional Poliomyelitis Laboratory Network, developed to support the program to eradicate indigenous wild poliovirus transmission in the Americas, included 10 laboratories, distributed in eight countries in the Americas, organized according to the diagnostic procedures they regularly performed. All laboratories isolated and typed virus in stool specimens, several did intratypic differentiation by nucleic acid probe hybridization, and 2 sequenced wild poliovirus isolates for molecular epidemiologic studies. High performance of the network was maintained through comprehensive training of virologists, continuous monitoring of laboratory performance, and prompt investigation of problems. Recommended field and laboratory procedures were regularly reviewed and revised to optimize sensitivity, specificity, and diagnostic efficiency. Close integration of field and laboratory surveillance was achieved through frequent meetings between virologists and epidemiologists, effective communication of program priorities, and the distribution of weekly surveillance reports.

Outbreak of poliomyelitis in Gizan, Saudi Arabia: cocirculation of wild type 1 polioviruses from three separate origins.

In 1989, a localized outbreak of 10 cases of poliomyelitis occurred in Saudi Arabia. Wild poliovirus type 1 was isolated from 5 patients. To determine the patterns of poliovirus circulation, partial nucleotide sequences of the poliovirus isolates were compared. These isolates were remarkably diverse. Two isolates were closely related to each other and to viruses isolated during the 1988 epidemic in Oman. Two other isolates were very similar to viruses found in Egypt. The fifth isolate was distantly related to the latter pair. The molecular data suggest that the 10 cases represented three separate outbreaks. The virologic findings underscore the potential for Saudi Arabia, which receives millions of guest workers and their families each year from countries in which polio is endemic, to be exposed to frequent importations of wild polioviruses. To restrict the circulation of imported polioviruses, Saudi Arabia must maintain high population immunity to poliovirus in all geopolitical divisions.

Molecular detection of an importation of type 3 wild poliovirus into Canada from The Netherlands in 1993.

During the fall and winter of 1992-1993 an outbreak of wild poliovirus type 3-associated poliomyelitis involving 71 patients occurred in The Netherlands. Almost all of the individuals involved in the outbreak belonged to an orthodox religious denomination that prohibits vaccination. A surveillance was initiated to determine if there had been an importation of this same strain of wild poliovirus into a southern Alberta community with a similar religious affiliation. Viral culture of stool samples from consenting individuals in the community resulted in viral isolates which typed as poliovirus type 3. Sequencing of amplicons generated from both the 5' nontranslated region and the VP1/2A portion of the genomes from representative poliovirus isolates indicated a greater than 99% genetic similarity to the strain from The Netherlands. The results of this study show that the utilization of PCR-based diagnostics offers an important molecular tool for the concise and rapid surveillance of possible cases of wild poliovirus importation into communities with individuals at risk for infection.

Group-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residue at positions of codon degeneracy.

We have developed a method for differentiating polioviruses from nonpolio enteroviruses using PCR. A pair of panpoliovirus PCR primers were designed to match intervals encoding amino acid sequences within VP1 that are strongly conserved among polioviruses. The initiating primer hybridizes with codons of a 7-amino-acid sequence that has been found only in polioviruses; the second primer matches codons of a domain thought to interact with the cell receptor. The panpoliovirus PCR primers contain mixed-base and deoxyinosine residues to compensate for the high degeneracy of the targeted codons. All RNAs from 48 vaccine-related and 110 wild poliovirus isolates of all three serotypes served as efficient templates for amplification of 79-bp product. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions. Sensitivities of poliovirus detection were as low as 100 fg (equivalent to approximately 25,000 genomic copies or 25 to 250 PFU) when the amplified products were visualized by ethidium bromide fluorescence. These degenerate PCR primers should aid in the detection of all polioviruses, including those wild poliovirus isolates for which genotype-specific reagents are unavailable.

Molecular characterization of a wild poliovirus type 3 epidemic in The Netherlands (1992 and 1993).

An outbreak of poliomyelitis due to wild poliovirus type 3 (PV3) occurred in an unvaccinated community in The Netherlands between September 1992 and February 1993. The outbreak involved 71 patients. The aim of this study was to characterize the virus at the molecular level and to analyze the molecular evolution of the epidemic virus. Molecular analysis was carried out by sequencing the VP1/2A junction region (150 nucleotides) of 50 PV3 strains isolated in association with this outbreak and the entire VP1 gene of 14 strains. In addition, the sequence of the VP1/2A junction region of strains from geographical regions endemic for PV3 (Egypt, India, and Central Asia) was analyzed and compared with the nucleotide sequence of the epidemic strain from The Netherlands. The earliest isolate was obtained from river water sampled 3 weeks before diagnosis of the first poliomyelitis patient and was found by VP1/2A sequence analysis to be genetically identical to the strain isolated from the first patient. Sequence divergence among the strains from the epidemic in The Netherlands was less than 2%. The closest genetic similarity (97.3%) was found with an Indian isolate (New Delhi, December 1991), indicating the likely source of the virus. A more than 99% sequence similarity was found in the VP1/2A region. Finally, the sequence information was used to design primers for the specific and highly sensitive molecular detection of PV3 strains during the epidemic.

Comparative study of five methods for intratypic differentiation of polioviruses.

A coded panel of 90 poliovirus isolates, 30 of each of the three known serotypes, was used to evaluate five methods for the intratypic differentiation of polioviruses: (i) an enzyme-linked immunosorbent assay with polyclonal cross-absorbed antisera (PAb-E), (ii) a neutralization assay with type-specific monoclonal antibodies (MAb-N), (iii) a restriction fragment length polymorphism (RFLP) assay, (iv) a Sabin vaccine strain-specific PCR assay, and (v) a Sabin vaccine strain-specific cRNA probe hybridization (ProHyb) assay. Sequence analysis was used for the definitive characterization of the strains. The panel was distributed to five laboratories; each laboratory analyzed the strains by at least two methods. Each method was used by three or four laboratories. The total performance scores (percentage correct results per number of tests) of the five methods were 96.7% for PAb-E, 93.9% for MAb-N, 91.9% for RFLP assay, 93.3% for Sabin vaccine strain-specific PCR, and 97.4% for Sabin vaccine strain-specific ProHyb. Consistent results were obtained by each laboratory for 88 of 90 isolates (97.8%) examined by PAb-E, 81 of 90 isolates (90.0%) examined by MAb-N, 78 of 90 isolates (86.7%) examined by RFLP assay, 81 of 90 isolates (90.0%) examined by PCR, and 89 of 90 isolates (98.9%) examined by ProHyb assay. Six strains were classified differently by different methods. It is recommended that at least two methods be used for the intratypic differentiation of poliovirus isolates, and each method should be based on a different principle (i.e., antigenic properties and nucleotide sequence composition). If two assays yield discrepant results, further characterization, preferably by partial sequence determination, will be required for correct identification.