Vaccine-related Polioviruses Research Articles

Wild poliovirus circulation among healthy children immunized with oral polio vaccine in Antananarivo, Madagascar.

From July 1995 to December 1996, 3185 stool specimens from healthy children aged 6-59 months attending 6 dispensaries in the Antananarivo area were examined for poliovirus. The children had been routinely immunized according to the Expanded Programme on Immunization (EPI) schedule and received the last dose of oral polio vaccine (OPV) more than 1 month before stool collection. 99.4% of the children were immunized with at least 3 doses of OPV. HEp-2 cell culture revealed virus infections in 192 stools (6.0%), including 9 poliovirus (0.3%) and 183 nonpolio enterovirus isolates (5.7%). Infections occurred throughout the year, but incidence was higher during the hot and rainy season (P=0.01). Using a neutralization test with monoclonal antibodies and PCR-RFLP in two genomic regions coding for the VP1 capsid and RNA polymerase, 4 wild polioviruses (3 type 1 and 1 type 3) and 5 vaccine-related polioviruses (2 Sabin 1-like variants, 1 Sabin 2-like and 2 Sabin 3-like) strains were identified. The wild polioviruses were isolated at the beginning and the end of the dry season. Similar RFLP patterns were observed for the 3 wild type 1 polioviruses. Comparison of partial genomic sequences in the VP1/2 A region of 1 of the wild type 1 isolates with 2 wild type strains isolated in Antananarivo in 1992 and 1993 showed a divergence of at least 10% between the strains, suggesting at least two different pathways of transmission during this period. Our findings demonstrate that immunization with 3 doses of OPV did not prevent intestinal carriage of wild poliovirus strains, and that there is a risk of wild poliovirus transmission to susceptible children in the area. Multiple strategies are required to improve immunization coverage in Madagascar.

Differentiation between vaccine-related and wild-type polioviruses using a heteroduplex mobility assay.

A heteroduplex mobility assay (HMA) was developed for intratypic differentiation between poliovirus isolates. The assay is based on polymerase chain reaction (PCR) amplification of a 480 base pair fragment which encodes a variable segment of VP1, followed by denaturation and reannealing of the resulting single strands with those from reference Sabin targets. Mismatches between wild-type and Sabin vaccine templates result in the formation of detectable heteroduplexes of reduced electrophoretic mobility. Poliovirus strains confirmed previously as wild-type or vaccine-like by PCR and sequencing were all correctly identified using the HMA. Mixtures of both wild-type and vaccine-like strains in a single isolate could also be detected using this technique. The results of this study demonstrate that heteroduplex analysis is a simple, rapid, and sensitive means for differentiating between vaccine-like and wild-type poliovirus isolates.

Identification of vaccine-related polioviruses by hybridization with specific RNA probes

We developed RNA probes for the identification of poliovirus isolates by blot hybridization. Two sets of vaccine strain-specific probes were prepared. They complemented variable genomic domains within (i) the 5'-untranslated region and (ii) the amino-terminal codons of VP1. An enterovirus group probe (EV/5UT) matching highly conserved 5'-untranslated region sequences was used to estimate the quantities of poliovirus (or enterovirus) RNA in the samples. Poliovirus sequences amplified from Sabin strain virion RNA templates by PCR were inserted into the pUC18 plasmid vector. The antisense PCR primer for each probe set contained sequences encoding a T7 promoter. Hybrids were detected by a sensitive nonisotopic method. RNA probes were labeled by incorporation of digoxigenin-uridylate into the transcripts. The binding of probe to immobilized poliovirus RNAs was visualized by hydrolysis of the chemiluminescent substrate 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2'-adamant ane) catalyzed by alkaline phosphatase conjugated to anti-digoxigenin (Fab) fragments. The specificities of the probes were evaluated with a panel of poliovirus isolates that had previously been characterized by sequence analysis. The RNAs of vaccine-related isolates hybridized with the appropriate probe sets. Wild polioviruses representing a broad spectrum of contemporary genotypes were recognized by the inabilities of their genomes to form stable hybrids with the Sabin strain-specific probes.

Direct detection of wild poliovirus circulation by stool surveys of healthy children and analysis of community wastewater.

Cartagena, Colombia, was one of the last cities in the Americas known to have endemic poliomyelitis. After 3 cases were identified in 1991, two approaches for detecting continued silent transmission of wild polioviruses within a high-risk community were used: stool surveys of healthy children and virologic analysis of community sewage. Wild type 1 polioviruses were isolated from 8% of the children studied and from 21% of sewage samples. The proportions of wild polioviruses, vaccine-related polioviruses, and nonpolio enteric viruses were similar for both approaches. Wild poliovirus sequences were also amplified directly from processed sewage samples by the polymerase chain reaction using primer pairs specific for the indigenous type 1 genotype. The last reported cases associated with wild polioviruses in the Americas occurred in Colombia (8 April 1991) and Peru (23 August 1991). Direct sampling for wild polioviruses in high-risk communities can provide further evidence that eradication of the indigenous wild polioviruses has been achieved in the Americas.

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 a 1b) 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 (> 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.

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 ≤ 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.

Viruses in sewage waters during and after a poliomyelitis outbreak and subsequent nationwide oral poliovirus vaccination campaign in Finland.

During an outbreak of paralytic poliomyelitis in Finland in 1984 and 1985 the widespread circulation of the causative wild-type serotype 3 poliovirus in the population was documented by demonstrating the virus in sewage water specimens in 13 different locations in the greater Helsinki district and in 13 other cities or towns all over the country. After the nationwide campaign with oral poliovirus vaccine in 1985, poliovirus serotypes 2 and 3 were readily isolated from sewage waters for up to 2 months, whereas type 1 poliovirus seemed to disappear from the sewage more rapidly. All of these isolates were temperature sensitive and therefore most likely vaccine related. The efficacy of the vaccination campaign in regard to elimination of the epidemic type 3 strain was evaluated by a follow-up study on viruses in sewage waters continued for 12 months through the subsequent expected season of poliomyelitis. Several types of enteroviruses, including five vaccine-related poliovirus strains, were identified in the 72 virus-positive specimens out of 93 studied. No wild-type polioviruses were found, indicating the success of the campaign.

Neurologic complications in oral polio vaccine recipients

Between April 1982 and June 1983 four children 3 to 24 months of age were referred for evaluation of neurologic abnormalities found to be compatible with vaccine-related poliovirus infection, which had not been suspected by referring physicians. Patients were epidemiologically unrelated residents of Indiana, and none had prior symptoms suggestive of immunodeficiency. All had received poliovirus vaccine orally (first dose in three, fourth dose in one) and a diphtheria-tetanus-pertussis injection in the left anterior thigh within 30 days of symptoms. A vaccine-like strain of poliovirus was isolated from each patient, and each had symptoms (left leg paralysis in three; developmental regression, spasticity, and progressive fatal cerebral atrophy in one) persisting for at least 6 months. Immune function was normal in two with poliovirus type 3 infection, and abnormal (hypogammaglobulinemia, combined immunodeficiency) in two with type 1 and type 2 infection, respectively. The incidence of observed vaccine-related poliovirus infection in Indiana recipients of orally administered poliovirus vaccine was 0.058 per 100,000 per year, significantly greater (P less than 0.001) than predicted.

Neurologic complications in oral polio vaccine recipients

In the period 1969-81, the CDC reported 37 cases of paralysis compatible with vaccine related poliovirus (VPV) infection in OPV recipients in the U.S.; no cases were reported in Indiana (pop. 5.5 million). From April 1982 to June 1983 we evaluated 4 children 3-24 mos of age whose neurologic abnormalities were compatible with VPV infection. All were epidemiologically unrelated residents of Indiana without symptoms suggestive of immunodeficiency. VPV infection was unsuspected prior to referral. All had OPV (1st dose in 3, 4th dose in 1) with DPT in the left anterior thigh within 30 days of symptoms, had VPV (confirmed by CDC) isolated at presentation, and had symptoms (left leg paralysis in 3, developmental regression, spasticity, and progressive fatal cerebral atrophy in 1) persisting for at least 6 mos. Immune function was normal in 2 with poliovirus type 3 infection and abnormal (hypogammaglobulinemia, combined immunodeficiency) in 2 with type 1 and type 2 infection. The incidence of observed VPV infection in Indiana OPV recipients was .058/100,000/yr, significantly greater (p = .02) than predicted. Neurologic complications among OPV recipients may: 1) Be increasing in frequency; 2) occur more frequently than estimated; 3) be incompletely reported; or 4) be more accurately detected with active surveillance.

Molecular variation of type 1 vaccine-related and wild polioviruses during replication in humans

Molecular variation in clinical isolates of type 1 poliovirus was followed at the level of the genome by oligonucleotide mapping and at the level of the gene products by polyacrylamide gel electrophoresis of the viral proteins. Oligonucleotide maps of epidemiologically distinct isolates were very different. In contrast, isolates from the recent Netherlands-Canada-USA poliomyelitis epidemic displayed very similar maps. Small but reproducible map changes were consistently observed for consecutive case isolates, which revealed the continual mutation and selection of the epidemic virus during replication in humans. Direct comparison of the maps of the first (Netherlands, 5/78) and the last (USA 6/79) epidemic case isolates suggested that about 100 base changes were fixed into the average viral genome during 13 months of epidemic transmission. Isolates from two recent vaccinees and one epidemiologically unrecognized contact similarly revealed small map differences from each other and from the pattern characteristic of the type 1 oral vaccine. Accompanying the genome changes were alterations in the mobilities and cleavage rates of the viral proteins. For both vaccine-related and wild isolates, the greatest variabilities were detected in the virion surface proteins, a pattern of change likely to lead to antigenic drift of the virus. Oligonucleotide mapping offers an independent and definitive approach for the recognition of genetic and epidemiologic relationships between poliovirus isolates.