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.

Genetic heterogeneity of the attachment glycoprotein G among group A respiratory syncytial viruses.

Fifteen independent group A respiratory syncytial virus (RSV) isolates were compared by sequencing a 300-nucleotide interval encoding a variable region of the attachment glycoprotein G. The viruses compared included the reference strains Long (USA 1956), A2 (Australia 1961), and 669 (Sweden 1959), along with 13 clinical isolates obtained at different times and locations throughout the United States. Representatives of all six antigenic subgroups, recognized by reactivity patterns with monoclonal antibodies, were compared. The maximum sequence heterogeneity within the G glycoprotein region compared was 15.7% of nucleotide sequences and 26% of amino acid sequences, more than twice the difference observed between Long and A2. Half of the nucleotide changes encoded amino acid substitutions, possibly indicating that the protein interval compared was subject to immune selection. Because the ratio of nucleotide to amino acid substitutions was nearly constant for all degrees of genetic divergence, the potential range of sequence divergence among group A RSV has probably not yet been attained. There was little correlation between the patterns of reactivity against a panel of monoclonal antibodies and sequence relationships among the 15 isolates. The sequence information showed multiple genotypes circulating simultaneously in the same community and very similar genotypes circulating in widely separated communities and during different years. Genetic analyses of RSV strains can provide important information about the relationships between RSV infections.

Detection and identification of vaccine-related polioviruses by the polymerase chain reaction.

We have used the polymerase chain reaction (PCR) to obtain sensitive detection and identification of poliovirus RNA genomes. Primer pairs were designed to permit identification of each Sabin poliovaccine strain by the electrophoretic mobilities of the amplified DNA products (Sabin 1: 97 bp; Sabin 2: 71 bp; Sabin 3: 44 bp). The compositions of samples containing mixtures of vaccine strains could be readily determined by PCR. When the amplified products were visualized by ethidium bromide fluorescence, as few as 250 genomic copies in the original sample could be detected. When PCR was used in combination with strain-specific 32P-labeled oligonucleotide probes, the limit of detection was less than or equal to 2.5 poliovirus genomes, exceeding the sensitivity of poliovirus isolation in cell culture by at least 100-fold. PCR amplifications may be performed on virion RNAs extracted directly from clinical specimens, potentially eliminating the requirement for virus isolation in routine identifications while yielding reliable results within 8 h.

Coupled mutations in the 5'-untranslated region of the Sabin poliovirus strains during in vivo passages: structural and functional implications.

All entero- and rhinovirus RNAs sequenced thus far possess A and U residues at positions corresponding to nucleotides 480 and 525, respectively, of poliovirus type 1. These two nucleotides have been proposed previously to form a base pair. The single exception to this rule appears to be the Sabin type 1 strain, which has a G480. Isolates of the Sabin 1 virus from healthy vaccinees were shown to have either a reversion to A480 or a second-site mutation U525----C, both restoring a potential for efficient base pairing. In vitro translation experiments demonstrated that poliovirus type 1 RNAs with either A480-U525 or G480-C525 are more efficient in promoting translation initiation as compared with the Sabin 1 RNA (G480-U525). The Sabin 2 strain has a U and an A at position 398 and 481, respectively, while its predecessor, strain P712, is shown to have C398 and G481. All the derivatives of the Sabin 2 isolated from vaccine-associated paralytic poliomyelitis cases shown reversion to G481, and most of them reverted also to C398. It is proposed that bases at positions 398 and 481 may be involved in a tertiary interaction. The in vitro template activity of the Sabin type 2 RNA (A481) is significantly lower than that of the isolate RNAs with G481, thus confirming the relation between attenuation and translation efficiency demonstrated previously for the type 1 and type 3 Sabin strains. The C----U change at position 398 exerted only a minor effect on the RNA template activity.

Genotype-specific in vitro amplification of sequences of the wild type 3 polioviruses from Mexico and Guatemala.

The extensive nucleotide sequence heterogeneity among independent genotypes of wild polioviruses permits the systematic design of genotype-specific molecular reagents. We have prepared two sets of polymerase chain reaction (PCR) primer pairs specific for the genotype of wild poliovirus type 3 recently endemic to Mexico and Guatemala. Nucleotide sequences of a representative wild type 3 virus isolated in Mexico in 1989 differed from the corresponding Sabin 3 (Leon 12 a1b) sequences at 167 of 900 positions within the VP1 region. From the sequence data, wild virus-specific primer pairs were designed to complement regions of high mismatch (greater than 33%) with Sabin 3 templates. Primer binding sites were spaced along the genome so that the predicted amplification products (142 bp and 163 bp) could be easily resolved electrophoretically from the products generated with our Sabin strain-specific primers (Sabin 1: 97 bp; Sabin 2: 71 bp; Sabin 3: 53 bp). RNAs of all wild type 3 poliovirus isolates from Mexico and Guatemala obtained over a 13-year period (1977-1990) served as efficient templates for amplification of the 142-bp and 163-bp products. Genomic templates derived from vaccine-related polioviruses and most heterologous wild polioviruses were inactive under equivalent reaction conditions. Amplifications generating a 114-bp product with a broadly reacting primer pair, matching highly conserved sequences in the 5'-noncoding region, provided a positive control for the presence in samples of poliovirus (or enterovirus) RNAs. Selective amplification of wild Mexico-Guatemala type 3 poliovirus sequences was obtained with either primer set in reactions containing large stoichiometric excesses (up to 10(6)-fold) of vaccine-related RNAs. We have used wild genotype-specific PCR primer sets to facilitate identification of wild polioviruses present in both clinical and environmental samples.

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.

Nucleotide sequences of the VP1 capsid proteins of wild poliovirus types 1 and 3 from epidemic areas of Brazil.

The nucleotide sequences encoding the capsid protein VP1 were determined for the wild polioviruses of serotypes 1 and 3 endemic to the northeastern region of Brazil. Compared with the corresponding Sabin vaccine strain sequences, the wild isolates differed at 20% (type 1) and 22% (type 3) of their nucleotide positions, and in 7% (type 1) and 11% (type 3) of their amino acid residues. The highest degree of amino acid heterogeneity occurred within the amino-terminal residues of the VP1 proteins. Intratypic amino acid differences also occurred in VP1 surface residues that form parts of antigenic sites for neutralizing antibodies.

Genetic co-regulation of galactose and melibiose utilization in Saccharomyces.

The gal3 mutation of Saccharomyces, which is associated with an impairment in the utilization of galactose, has been shown to be pleiotropic, causing similar impairments in the utilization of melibiose and maltose. Milibiose utilization and alpha-galactosidase production are directly controlled by the galactose regulatory elements i, c, and GAL4. The fermentation of maltose and the induction of alpha-glucosidase are regulated independently of the i, c, GAL4 system. The production of alpha-galactosidase and galactose-1-phosphate uridyl transferase is coordinate in galactokinaseless strains. Galactose serves as a nonmetabolized, gratuitous inducer of alpha-galactosidase in strains lacking the genes for one or more of the Leloir pathway enzymes.

Molecular epidemiology of polioviruses.

Poliovirus isolates can be identified according to their genotypes with use of the technique of oligonucleotide fingerprinting. Fingerprint analysis is performed by cleaving the viral RNA genome with ribonuclease T1 and separating the fragments (oligonucleotides) in two dimensions. The larger, structurally unique oligonucleotides distribute into patterns ("fingerprints") highly characteristic of a specific overall RNA sequence. Isolates from the same epidemic have very similar fingerprints. Isolates from distinct epidemics have very different fingerprints, a consequence of the rapid evolution of polioviruses during replication in humans. Similarity in the fingerprints of case isolates provides independent evidence for epidemiologic linkage. Fingerprinting can readily distinguish vaccine-related isolates from wild strains. Contemporary vaccine-related isolates are very probably vaccine-derived because their fingerprints contain characteristic vaccine-strain oligonucleotide spots (types 1 and 3) and because their wild-type parents are unlikely to have survived largely unaltered in the natural environment. Some examples of applications of this technique within different epidemiologic settings are described.

Monoclonal antibodies of four different specificities for neutralization of type 1 polioviruses.

Four independent hybridoma clones have been established that produce neutralizing antibodies specific to type 1 poliovirus. Each clone produced antibody which neutralized a distinct set of type 1 test strains: (i) all 15 strains tested; (ii) the inducer strain only; (iii) predominantly wild strains; or (iv) all vaccine-related and some wild strains.

Regions of poliovirus protein VP1 produced in Escherichia coli induce neutralizing antibodies.

Poliovirus type 1 cDNA was prepared from viral RNA encoding the VP1 capsid region of the virus by using a specific DNA primer and was cloned in Escherichia coli. DNA fragments corresponding to VP1 amino acid positions 129 to 302 (pPM5k3), 52 to 302 (pPMhae3), and 24 to 129 (pPMDxba) were incorporated into plasmid vectors designed to express Trp LE-poliovirus VP1 fusion proteins under the control of the inducible tryptophan promoter-operator system. Induction of bacterial cultures containing the plasmids resulted in the production of fusion proteins which accounted for 21% (pPMhae3), 68% (pPM5k3), and 27% (pPMDxba) of the total cell protein. The proteins were purified, and each reacted with polyclonal antibodies raised against intact virions as measured by an enzyme-linked immunosorbent assay. The sera from rabbits immunized with the bacterially produced fusion proteins pPMDxba and pPMhae3 contained poliovirus-neutralizing antibodies.

Reemergence of an epidemic coxsackievirus B5 genotype.

Outbreaks of coxsackievirus B5 (CB5) infections occur primarily during peak epidemic years, with comparatively few cases occurring during intervening years. This pattern of periodic CB5 epidemicity is quite distinct from the general endemicity typical of other group B coxsackieviruses. To determine the genetic relationships among CB5 isolates from different outbreaks, we compared viral RNAs by ribonuclease T1 oligonucleotide fingerprinting. Isolates obtained within an epidemic year had very similar fingerprints, an observation indicating that they were closely related variants of a single genotype. CB5 isolates from the major 1972 epidemic were not closely related to the genotype associated with the preceding epidemic of 1967. However, isolates from the most recent CB5 epidemic year, 1983, had fingerprints nearly identical to those of the 1967 strains. These findings provide clear evidence for epidemic reemergence of the 1967 genotype and suggest that the virus was maintained under conditions approaching evolutionary stasis during the intervening 16-year period.

Changes in three of the four coat proteins of oral polio vaccine strain derived from type 1 poliovirus.

Little is yet known about the nature, or extent, of the changes involved in attenuation of neurovirulent poliovirus. The tryptic comparison reported here, of coat proteins from the Sabin type 1 polio vaccine and parental Mahoney virus, provides a useful approach and affords some insight into this question. The main obstacle, separation of the labile proteins VP1 and VP2 in an intact state from the vaccine strain, was overcome by incorporating 3.5 M urea into an otherwise standard preparative gel electrophoresis system. Tryptic maps revealed six altered leucine-containing peaks: two in VP1, none in VP2, three in VP3, and one in VP4. It is estimated, after correcting for leucine-free peptides, that the coat protein sequences may have undergone some 10 to 13 amino acid replacements, roughly 1.5% of the total, in the course of attenuation leading to the vaccine strain.

Geographic distribution of wild poliovirus type 1 genotypes.

Determination of the patterns of genomic variation among RNA virus isolates is a powerful approach for establishing their epidemiologic interrelationships. The standard technique for such studies, ribonuclease T1 oligonucleotide fingerprinting, can detect similarities only among very closely related isolates. The rapid evolution of the poliovirus genome during transmission in humans requires the application of alternate methods to identify more distant relationships. To obtain a substantially broader view of the distribution of wild poliovirus type 1 genotypes in nature, we compared 150 bases of genomic sequence information (encoding parts of the capsid protein VP1 and the noncapsid protein 2A) from 62 isolates obtained from poliomyelitis patients in five continents. The partial sequence information allowed us to (1) identify numerous geographic foci of endemic circulation of wild type 1 polioviruses, (2) reveal previously unsuspected links between cases in distant communities, (3) monitor the displacement from the environment of preexisting polioviruses by viruses from other regions, and (4) recognize the recombinant (vaccine-wild; wild-wild) origins of some epidemic polioviruses.

Natural variants of the Sabin type 1 vaccine strain of poliovirus and correlation with a poliovirus neutralization site.

Independent substitution mutations have been detected in capsid polypeptide VP1 of the type 1 oral poliovirus vaccine isolated from normal infant vaccine recipients. These mutations map at amino acid residues 142 and 147 of VP1, a region only minimally hydrophilic. A synthetic peptide, corresponding to residues 141 to 147 of VP1 was synthesized, conjugated to a carrier polypeptide of bovine serum albumin. The conjugate was found to elicit a weak poliovirus neutralizing antibody response. It was also capable of priming the immune system for the production of IgG-type antibodies able to neutralize greater than 99.999% of infectious type 1 virus. It is suggested that region 141 to 147 of VP1 may be involved in neutralization of the virus and that the mutants may have accumulated by antibody selection.

Application of the polymerase chain reaction (PCR) to poliomyelitis surveillance through the analyses of sewage samples.

A rapid and sensitive method for the detection of wild poliovirus from sewage samples using the polymerase chain reaction (PCR) technique was investigated. To eliminate the toxicity of sample concentrates to the enzymatic system used in PCR, a methodology was developed for the purification of these concentrates, consisting of treatment with trichlorofluoroethane and Sephadex column chromatography. The viral RNA was extracted from the purified concentrates, submitted to PCR with primers specific for Brazilian wild poliovirus type 1 and for Sabin types 1, 2 and 3. The amplified products were detected by electrophoresis in vertical polyacrylamide gels and stained with ethidium bromide. The results suggest that sewage sampling for environmental surveillance, combined with the rapid and precise PCR technology, provides a powerful tool for assessment of the success of the poliovirus eradication programme.

Multiple genetic changes can occur in the oral poliovaccines upon replication in humans.

Poliovirus isolates of serotypes 2 and 3 from patients whose paralytic poliomyelitis cases were classified as oral vaccine-associated were analysed by oligonucleotide mapping of the virus genomes and by polyacrylamide gel electrophoresis of the virus proteins. Oligonucleotide maps of all isolates were similar to the maps of the corresponding oral vaccine strain. No two isolates gave identical maps. Most maps differed from that of the vaccine strain by at least one oligonucleotide spot. Maps of some isolates revealed numerous differences, indicating that multiple (greater than 100) genetic changes had occurred in the vaccine virus genomes during replication in one or two individuals. In contrast, maps of some neural tissue isolates showed minimal differences from the reference vaccine maps, raising the possibility that neurovirulence may be restored by a small number of genetic changes. For many isolates, changes were also detected in the mobilities of processing rates of the virus proteins.

Strain characterization studies of poliovirus type I isolates from poliomyelitis cases in the United States in 1979.

In 1979 there was a small outbreak of poliomyelitis among non-immunized Amish individuals in the United States. Epidemiological evidence linked these cases to those in Canada in 1978 which themselves were related to cases in the Netherlands in 1978. Poliovirus type 1 was found to be the cause both in the US and in the other countries. Strain characterization tests were carried out on virus isolates from all three outbreaks. All the type 1 strains were non-vaccine-like both with the absorbed strain specific antisera and with the modified Wecker test. However, the latter as well as the rct test used to characterize these strains are obviously not very discriminating marker tests. The oligonucleotide mapping procedure appears to be a much more definitive approach to recognizing relationships between polioviruses. The various marker tests provided laboratory data in support of the epidemiological evidence that the poliovirus type 1 spread from Holland to Canada in 1978 and from there to the US in 1979.

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.