Circulating vaccine-derived polioviruses in the Extreme North region of Cameroon

BackgroundThe World Health Organization (WHO) poliovirus eradication program includes careful surveillance of acute-flaccid paralysis (AFP) and mass and routine immunization with oral polio vaccine (OPV). In populations with low vaccine coverage, the live-attenuated Sabin strains, OPV types 1, 2 and 3, can evolve into virulent vaccine-derived polioviruses (VDPVs) and circulate in the community. Until recently, circulating VDPVs (cVDPVs) had not been reported in Cameroon despite the fact that VDPV2 outbreaks have occurred in nearby countries.ObjectivesThis study aimed to characterize virus isolates from four AFP patients infected with cVDPV2 in the Extreme North region of Cameroon in 2013.Study designThe complete VP1 region of the four VDPV strains was sequenced and the relationships with cVDPVs from neighboring countries were investigated.ResultsAll four patients were infected by cVDPV2 strains showing 1.2–2.0% nucleotide difference compared to the reference Sabin 2 VP1 sequence. Phylogenetic analysis indicated that the VDPV strains were genetically linked to cVDPV2 lineages of the recent Chad cVDPV2 outbreak.ConclusionsThe circulation of pathogenic VDPVs suggests that there are localized immunization gaps in some districts like Makary, Mada and Kolofata in Cameroon. To avoid poliomyelitis outbreaks in Cameroon, especially in the districts close to neighboring countries with ongoing cVDPV outbreaks, high polio vaccine coverage is essential.     

Circulation of a type 1 recombinant vaccine-derived poliovirus strain in a limited area in Romania

Summary After intensive immunisation campaigns with the oral polio vaccine (OPV) as part of the Global Polio Eradication Initiative, poliomyelitis due to wild viruses has disappeared from most parts of the world, including Europe. Here, we report the characterization of a serotype 1 vaccine-derived poliovirus (VDPV) isolated from one acute flaccid paralysis (AFP) case with tetraplegia and eight healthy contacts belonging to the same small socio-cultural group having a low vaccine coverage living in a small town in Romania. The genomes of the isolated strains appeared to be tripartite type 1/type 2/type 1 vaccine intertypic recombinant genomes derived from a common ancestor strain. The presence of 1.2% nucleotide substitutions in the VP1 capsid protein coding region of most of the strains indicated a circulation time of about 14 months. These VDPVs were thermoresistant and, in transgenic mice expressing the human poliovirus receptor, appeared to have lost the attenuated phenotype. These results suggest that small populations with low vaccine coverage living in globally well-vaccinated countries can be the origin of VDPV emergence and circulation. These results reaffirm the importance of active surveillance for acute flaccid paralysis and poliovirus in both polio-free and polio-endemic countries.     

A Sabin 2-related poliovirus recombinant contains a homologous sequence of human enterovirus species C in the viral polymerase coding region

A type 2 vaccine-related poliovirus (strain CHN3024), differing from the Sabin 2 strain by 0.44% in the VP1 coding region was isolated from a patient with vaccine-associated paralytic poliomyelitis. Sequences downstream of nucleotide position 6735 (3D ^pol coding region) were derived from an unidentified sequence; no close match for a potential parent was found, but it could be classified into a non-polio human enteroviruses species C (HEV-C) phylogeny. The virus differed antigenically from the parental Sabin strain, having an amino acid substitution in the neutralizing antigenic site 1. The similarity between CHN3024 and Sabin 2 sequences suggests that the recombination was recent; this is supported by the estimation that the initiating OPV dose was given only 36–75 days before sampling. The patient’s clinical manifestations, intratypic differentiation examination, and whole-genome sequencing showed that this recombinant exhibited characteristics of neurovirulent vaccine-derived polioviruses (VDPV), which may, thus, pose a potential threat to a polio-free world.     

Three cases of paralytic poliomyelitis associated with type 3 vaccine poliovirus strains in Bulgaria

Oral poliovirus vaccine (OPV) can cause, in extremely rare cases vaccine‐associated paralytic poliomyelitis in recipients, or contacts of vaccinees. Three cases of vaccine‐associated paralytic poliomyelitis (two contacts and one recipient) occurred in the Bourgas region of Bulgaria in the spring of 2006. The first two cases, notified as acute flaccid paralysis, were 55 days old unvaccinated twin brothers, having been in contact with vaccinees. The third case concerned a 4‐month‐old infant who had received the first OPV dose 37 days prior to the onset of illness. Complete clinical, epidemiological, virological, serological and molecular investigations of the children with paralysis and their contacts were undertaken. In all the three cases type 3 polioviruses were isolated from fecal samples and characterized as Sabin‐like poliovirus strains. Type 3 polioviruses isolated from the twin brothers demonstrated by sequence analysis U‐to‐C back mutation at nt 472 of the 5′ UTR, known to correlate with neurovirulence, and mutation in the VP1 region. Type 3 poliovirus isolated from the third child demonstrated in the 3D sequenced region a recombination with Sabin type 1 poliovirus. In the latter region, three silent mutations and one, resulting in amino acid substitution, were also observed. The clinical, epidemiological and virological data and the neurological sequelae observed 60 days following the onset of paralysis, confirmed the diagnosis of vaccine‐associated paralytic poliomyelitis in all the three patients. J. Med. Virol. 81:1661–1667, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Isolation of a type 3 vaccine-derived poliovirus (VDPV) from an Iranian child with X-linked agammaglobulinemia

Type 3 immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) were isolated from a 15-month-old Iranian boy with acute flaccid paralysis (AFP) who was subsequently diagnosed with X-linked agammaglobulinemia (XLA). VP1 nucleotide sequences of the two isolates differed from Sabin 3 by 2.0% and 2.1% and from each other by 0.6%. Although the key determinant of attenuation and temperature sensitivity in the 5'-untranslated region (U(472)-->C) had reverted, a second capsid-region determinant (VP3:Phe(091)) was unchanged, but a presumptive suppressor (VP1:Ala(054)-->Val) was found. The isolates were Sabin 3/Sabin 1 recombinants, sharing a single recombination breakpoint in the 2C region. Although the two isolates were antigenically distinct from Sabin 3, only one amino acid replacement was found in the neutralizing antigenic sites (VP3:Ser(059)-->Asn in site 3). The patient was placed on intravenous immunoglobulin (IVIG) therapy within 9 days of onset of AFP, and iVDPV excretion ceased thereafter, but the patient remained severely paralyzed until his death approximately 11 months after paralysis. No secondary AFP cases were found, and none of the seven tested contacts of the patient were found to be infected with poliovirus.     

Mutations in Sabin 2 strain of poliovirus and stability of attenuation phenotype.

In this study, we attempted to identify the molecular determinants in the genome of the attenuated Sabin 2 vaccine strain of poliovirus that may change during vaccine production and result in an increase in monkey neurovirulence. An extensive search for suitable vaccine lots identified six batches that had failed the monkey neurovirulence test (MNVT). On repeated tests, these batches were found to have acceptable levels of monkey neurovirulence. One of the batches was additionally passaged six times under conditions used in vaccine production, and the resulting high-passage sample was screened for the presence of mutations and tested in monkeys. In addition to the previously described A --> G reversion at nucleotide 481, high-passage stock also contained a mutation in the VP1-coding region (3364 = G --> A) that consistently accumulated in the course of passaging. However, despite the presence of substantial amounts of these mutations, high-passage stock passed the MNVT. Replication of Sabin 2 poliovirus in the central nervous system of transgenic mice susceptible to poliovirus or in cultures of mouse cells, resulted in another mutation (3363 = A --> G). Even though its presence correlated with paralysis in mice, the introduction of 3363-G into the Sabin 2 genome did not increase neurovirulence of the virus. Previous studies identified the 481-G mutation as an important determinant of monkey neurovirulence. We prepared virus samples with varying amounts of genetically defined single mutants at this nucleotide and tested them in monkeys. The results demonstrated that even a 100% substitution at this site introduced into Sabin 2 strain did not increase monkey neurovirulence. The determination of the nucleotide sequence of an alternative strain used for the production of type 2 OPV (Chung 2) showed that it contained 100% of the wild-type 481-G but possessed an extremely low level of neurovirulence. These results demonstrate the remarkable stability of the attenuated phenotype of the Sabin 2 strain and show that (1) no batch of OPV 2 has ever repeatedly failed the MNVT, (2) growing the virus beyond the passage level allowed in vaccine production did not result in increased neurovirulence in monkeys, (3) a test for neurovirulence in transgenic mice may be more sensitive than the MNVT, and (4) determination of the mutational profile of vaccine batches detects inconsistencies in vaccine manufacturing processing that would not be detected by the MNVT.     

Remarkable cross-reaction of pan-pestivirus PCR primers with poliovirus genome

When several human vaccines were tested for pestivirus contamination using a one-tube closed nested RT-PCR method employing pan-pestivirus primers selected from the 5'-untranslated region (5'-UTR) of the pestivirus genome, a 224 bp DNA product was produced from a poliovirus vaccine. Although this amplicon was of the size expected for pestiviruses, its sequence showed a 100% similarity with the corresponding reverse complement of a nucleotide sequence from the VP2 gene of the poliovirus type 1 Sabin strain. It is recommended that all positive PCR products, especially those prepared using pan-pestivirus primers, obtained from screening biological substances for pestivirus contamination should be checked by use of a specific hybridization probe and preferably by sequencing.     

Characterization of mutations in the VP(1) region of Sabin strain type 1 polioviruses isolated from vaccine-associated paralytic poliomyelitis cases in Iran.

BACKGROUND: The live-attenuated oral polio vaccine used to interrupt poliovirus transmission is genetically unstable. Reversion of some attenuating mutations, which normally occurs during vaccine strain replication in some recipients, and can rarely cause vaccine-associated paralytic poliomyelitis (VAPP). The poliovirus eradication program designed by the World Health Organization (WHO) includes immunization with OPV in addition to careful surveillance of all acute-flaccid paralysis (AFP) cases. OBJECTIVES: In Iran we last isolated imported wild poliovirus in 2000 and the immunization coverage was 100% in 2002. During 2001, there were three AFP cases with residual paralysis from which Sabin-like type 1 polioviruses were isolated in our national polio laboratory. STUDY DESIGN: The complete VP(1) region of the three isolates was sequenced and amino acid substitutions associated with these neurovirulent isolates were recorded. RESULTS: These isolates had either 4, 2 or 1 nucleotide substitution(s) in the VP(1) region, corresponding to amino acid change in the VP(1) of isolate 1 of either (H-[149]->Y), (T-[106]->A) or (I-[90]->L), respectively. CONCLUSIONS: Surveillance of the VAPP cases in countries where endemic transmission has recently ceased increases our understanding of the important neurovirulent mutations in vaccine-strain isolates and assists in planning the next step in the eradication program in these countries.     

Inactivated polio vaccine: Time to introduce it in India's national immunization schedule

Polio is a communicable disease caused by poliovirus that may attack nerve cells of the brain and spinal cord. The victims develop neurological complications, likes stiffness of the neck, muscular weakness, or paralysis of one or more limbs. In severe cases, it may be fatal due to respiratory paralysis. The world has seen tremendous gains in polio eradication over the past year. India and Nigeria saw a reduction in cases of almost 95% from 2009 to 2010, and cases of wild poliovirus type 3 (WPV3) fell by 92% globally over the same period. In fact, no case has been reported in India since February 2011, such that India may be on the verge of eradicating polio. Nevertheless, polio control experts are particularly worried about Vaccine-Derived Poliovirus (VDPV). Global surveillance efforts picked up 430 cases of VDPV from several countries between July 2009 and March 2011. In India, 7 cases of VDPV were reported during the year 2011. As long as OPV is used, virologists say that the world is at risk of VDPV causing polio in unprotected children. Achieving a polio-free world will require the "cessation of all OPV" and with it the elimination of the risk of vaccine-associated paralytic polio (VAPP) or VDPV infections. To this effect, in 2011 the Global Polio Eradication Initiative (GPEI) will produce and develop a new roadmap for VDPV Elimination. Several countries have shifted from all OPV to sequential OPV-IPV schedules and all-IPV schedules with elimination of live poliovirus. IPV will be indispensable in the post-eradication era when use of OPV has to stop but "vaccination against polio" cannot stop. IPV offers complete individual protection and has been considered as an additional tool at present for those who can afford the vaccine, and since we are nearing the eradication of polio, it is time to shift from OPV to sequential OPV-IPV schedule in India. Such a strategy will avoid inevitable problems with VAPP.     

Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.     

Genomic analysis of vaccine-derived poliovirus strains in stool specimens by combination of full-length PCR and oligonucleotide microarray hybridization

Sabin strains of poliovirus used in the manufacture of oral poliovirus vaccine (OPV) are prone to genetic variations that occur during growth in cell cultures and the organisms of vaccine recipients. Such derivative viruses often have increased neurovirulence and transmissibility, and in some cases they can reestablish chains of transmission in human populations. Monitoring for vaccine-derived polioviruses is an important part of the worldwide campaign to eradicate poliomyelitis. Analysis of vaccine-derived polioviruses requires, as a first step, their isolation in cell cultures, which takes significant time and may yield viral stocks that are not fully representative of the strains present in the original sample. Here we demonstrate that full-length viral cDNA can be PCR amplified directly from stool samples and immediately subjected to genomic analysis by oligonucleotide microarray hybridization and nucleotide sequencing. Most fecal samples from healthy children who received OPV were found to contain variants of Sabin vaccine viruses. Sequence changes in the 5' untranslated region were common, as were changes in the VP1-coding region, including changes in a major antigenic site. Analysis of stool samples taken from cases of acute flaccid paralysis revealed the presence of mixtures of recombinant polioviruses, in addition to the emergence of new sequence variants. Avoiding the need for cell culture isolation dramatically shortened the time needed for identification and analysis of vaccine-derived polioviruses and could be useful for preliminary screening of clinical samples. The amplified full-length viral cDNA can be archived and used to recover live virus for further virological studies.     

Molecular evolution and antigenic drift of type 3 iVDPVs excreted from a patient with immunodeficiency in Ningxia,China

A 2.5-year-old pediatric patient with acute flaccid paralysis was diagnosed with primary immunodeficiency (PID) in Ningxia Province, China, in 2011. Twelve consecutive stool specimens were collected from the patient over a period of 10 months (18 February 2011 to 20 November 2011), and 12 immunodeficiency vaccine-derived poliovirus (iVDPV) strains (CHN15017-1 to CHN15017-12) were subsequently isolated. Nucleotide sequencing analysis of the plaque-purified iVDPVs revealed 2%–3.5% VP1-region differences from their parental Sabin 3 strain. Full-length genome sequencing showed they were all Sabin 3/Sabin 1 recombinants, sharing a common 2C-region crossover site, and the two key determinants of attenuation (U472C in the 5′ untranslated region and T2493C in the VP1 region) had reverted. Temperature-sensitive experiments demonstrated that the first two iVDPV strains partially retained the temperature-sensitive phenotype's nature, while the subsequent ten iVDPV strains distinctly lost it, possibly associated with increased neurovirulence. Nineteen amino-acid substitutions were detected between 12 iVDPVs and the parental Sabin strain, of which only one (K1419R) was found on the subsequent 10 iVDPV isolates, suggesting this site's potential as a temperature-sensitive determination site. A Bayesian Monte Carlo Markov Chain phylogenetic analysis based on the P1 coding region yielded a mean iVDPV evolutionary rate of 1.02 × 10⁻² total substitutions/site/year, and the initial oral-polio-vaccine dose was presumably administered around June 2009. Our findings provide valuable information regarding the genetic structure, high-temperature growth sensitivity, and antigenic properties of iVDPVs following long-term evolution in a single PID patient, thus augmenting the currently limited knowledge regarding the dynamic changes and evolutionary pathway of iVDPV populations with PID during long-term global replication.     

Succession of Mutations in the Sabin Strain of Type 3 Poliovirus Replicating in the Central Nervous System of Monkeys

Sabin strains of oral poliovirus vaccine (OPV) undergo limited genetic changes during replication in cell cultures, the gastrointestinal tract of vaccinees, and the central nervous system of monkeys. Some of these changes are associated with loss of attenuation markers. Here we report the dynamics of mutant accumulation in the Sabin strain of poliovirus type 3 inoculated intraspinally into monkeys. Thr → Ile reversion in amino acid 6 of VP1 (2493 C → U) occurred within the first few days postinoculation (p.i.), but decreased on later days and completely disappeared by Day 17 p.i. 472 U → C reversion in the 5′-untranslated region appeared to accumulate slower and by Day 17 completely substituted for the vaccine-type nucleotide at this site. These results indicate that experimental infection of the central nervous system of monkeys consists of early and late phases in which a different genetic constitution of the virus is favored. In several isolates one additional neurovirulent revertant was found: a Phe → Ser at amino acid 91 of VP3 (2034 U → C). Since this mutation was never detected in vaccine lots and is strongly selected against in cell cultures at temperatures below 38.5°, it does not threaten the safety of OPV.     

Sabin Type 2 Polioviruses with Intertypic Vaccine/Vaccine Recombinant Genomes

Attenuated strains of the Sabin oral poliovirus vaccine replicate in the human gut and, in rare cases, cause vaccine-associated paralytic poliomyelitis. In the present study, 15 vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis and from healthy vaccinees were examined. Four distant sequences of the poliovirus genome (5^′ NCR, VP3/VP1, VP1/2A, and 3DPol/3^′ NCR) were targeted, and the reverse-transcribed segments were amplified by polymerase chain reaction followed by restriction fragment length polymorphism analysis with four restriction enzymes. Among the 15 isolates (11 Sabin type 2, 3 Sabin type 1, and 1 Sabin type 3), four Sabin type 2 isolates (36%) were found to be intertypic vaccine/vaccine recombinant in the 3DPol/3^′ NCR region of the viral genome. The recombinant genotypes identified were S2/S2/S1 for two isolates and S2/S2/S2/S3 and S2/S2/S1/S2 for each of the other two isolates, respectively. Recombinant viruses with unmodified segments in the 5^′ NCR and the VP3/VP1 regions of the viral genome, a modified segment in the VP1/2A region only for one strain, and an often recombinant segment in the 3DPol/3^′ NCR parts of the genome were so identified. These findings provide strong evidence that recombination is a frequent phenomenon in type 2 poliovirus vaccine strains and suggest that recombination may be an important mechanism of the natural evolution of polioviruses of Sabin type 2 origin, perhaps even one of the mechanisms of reversion of attenuated vaccine strains toward neurovirulence. Electronic Publication     

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 ~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 ~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 ~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.     

Retrospective molecular and phenotypic analysis of poliovirus vaccine strains isolated in Greece

The live oral poliovirus vaccine (OPV) strains are genetically unstable, causing, in rare cases, vaccine-associated paralytic poliomyelitis. Reversions of the known attenuating mutations in OPV strains and intertypic recombination have been identified as the underlying causes of the increased neurovirulence of poliovirus isolates. In this study, three OPV isolates (one non-recombinant and two recombinants) were tested in order to correlate phenotypic traits such as temperature sensitivity (Rct test) and growth kinetics (one-step growth curve test) with mutations and recombination events of the viral genome. Moreover, the immunity level of the western Greek population aged 1–40 years was evaluated against OPV isolates and Sabin vaccine strains, with a microneutralization assay. Members of the 1–40-year age group (both pooled and individual sera) showed no significant differences in neutralization test (NT) titres against OPV isolates in comparison with the Sabin vaccine strains. However, all three OPV isolates showed reverted phenotypic traits in Rct or one-step growth curve assays. The results of our study revealed a significant decrease in immunity level from the 1–10-year age group to the 21–30-year age group (pooled sera) for both poliovirus types 1 and 3. For both poliovirus types, the highest NT titres were observed in the 1–10-year age group, and the lowest NT titre was observed in the 21–30-year age group, towards poliovirus type 3. Our study underlines the need for immunological studies in all age groups, in order to allow reconsideration of the current vaccination policies and to avoid epidemics caused by the circulation of highly evolved OPV derivatives.     

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.     

Genetic basis of the neurovirulence of type 1 polioviruses isolated from vaccine-associated paralytic patients

Summary We examined four type 1 polioviruses isolated from the stools of patients with vaccine-associated paralytic poliomyelitis in China. All of these isolates were shown to be Sabin derived viruses by restriction fragment length polymorphism assay after polymerase chain reaction and by sequencing of the viral genome encoding the viral coat protein, VP1. However, the same analysis of the 3D coding region suggested that two of the four isolates had the sequence of wild type poliovirus in the tested region. Furthermore there were also point mutations in the 5 non-coding region. One was a single base change from U to C at nucleotide position 525, and the other three were from G to A at position 480. All the four strains were more neurovirulent than Sabin type 1 virus in transgenic mice with human poliovirus receptor gene. The data showed that the nucleotide positions of type 1 poliovirus which were identified to be in favor of the high neurovirulence were indeed changed during natural transmission, and suggested that the point mutation alone or a recombination of the vaccine type with wild type genome results in an acquisition of neurovirulence.     

Identification of candidate sequences that determine virulence in Coxsackievirus B4.

We have previously shown that a major determinant of virulence for coxsackievirus B4 mapped to the 5' end of the viral genome. Comparison of the corresponding cDNA sequences of a virulent and a non-virulent virus has allowed the identification of candidate determinants of virulence in the 5' untranslated region and the capsid proteins VP1, VP2 and VP4. Thirteen nucleotide substitutions were observed in a region spanning 3298 nucleotides. Four mutations were detected in the non-coding region. Of the remaining nine mutations, four were silent while five resulted in amino acid substitutions in VP1, VP2 and VP4. The amino acid substitutions in the virulent virus were analyzed in relation to the three-dimensional structures of the capsid proteins of poliovirus. Two substitutions mapped to the amino termini of VP1 and VP4. Of the two substitutions observed in VP2, one mapped to the large loop that connects beta strand E with the radial helix on the back surface of the eight-stranded antiparallel beta barrel while the other mapped to beta strand G. One amino acid substitution in VP1 mapped to the loop connecting beta strands D and E at a site close to a major determinant of attenuation in poliovirus type 2.