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

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

Circulation of type 1 vaccine-derived poliovirus in the Philippines in 2001

In 2001, highly evolved type 1 circulating vaccine-derived poliovirus (cVDPV) was isolated from three acute flaccid paralysis patients and one contact from three separate communities in the Philippines. Complete genomic sequencing of these four cVDPV isolates revealed that the capsid region was derived from the Sabin 1 vaccine strain but most of the noncapsid region was derived from an unidentified enterovirus unrelated to the oral poliovirus vaccine (OPV) strains. The sequences of the cVDPV isolates were closely related to each other, and the isolates had a common recombination site. Most of the genetic and biological properties of the cVDPV isolates were indistinguishable from those of wild polioviruses. However, the most recently identified cVDPV isolate from a healthy contact retained the temperature sensitivity and partial attenuation phenotypes. The sequence relationships among the isolates and Sabin 1 suggested that cVDPV originated from an OPV dose given in 1998 to 1999 and that cVDPV circulated along a narrow chain of transmission. Type 1 cVDPV was last detected in the Philippines in September 2001, and population immunity to polio was raised by extensive OPV campaigns in late 2001 and early 2002.     

Twenty-Eight Years of Poliovirus Replication in an Immunodeficient Individual: Impact on the Global Polio Eradication Initiative

There are currently huge efforts by the World Health Organization and partners to complete global polio eradication. With the significant decline in poliomyelitis cases due to wild poliovirus in recent years, rare cases related to the use of live-attenuated oral polio vaccine assume greater importance. Poliovirus strains in the oral vaccine are known to quickly revert to neurovirulent phenotype following replication in humans after immunisation. These strains can transmit from person to person leading to poliomyelitis outbreaks and can replicate for long periods of time in immunodeficient individuals leading to paralysis or chronic infection, with currently no effective treatment to stop excretion from these patients. Here, we describe an individual who has been excreting type 2 vaccine-derived poliovirus for twenty eight years as estimated by the molecular clock established with VP1 capsid gene nucleotide sequences of serial isolates. This represents by far the longest period of excretion described from such a patient who is the only identified individual known to be excreting highly evolved vaccine-derived poliovirus at present. Using a range of in vivo and in vitro assays we show that the viruses are very virulent, antigenically drifted and excreted at high titre suggesting that such chronic excreters pose an obvious risk to the eradication programme. Our results in virus neutralization assays with human sera and immunisation-challenge experiments using transgenic mice expressing the human poliovirus receptor indicate that while maintaining high immunisation coverage will likely confer protection against paralytic disease caused by these viruses, significant changes in immunisation strategies might be required to effectively stop their occurrence and potential widespread transmission. Eventually, new stable live-attenuated polio vaccines with no risk of reversion might be required to respond to any poliovirus isolation in the post-eradication era.     

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

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

Neurovirulence of type 1 polioviruses isolated from sewage in Japan.

Sixteen type 1 poliovirus strains were isolated from a sewage disposal plant located downstream of the Oyabe River in Japan between October 1993 and September 1995. The isolates were intratypically differentiated as vaccine-derived strains. Neutralizing antigenicity analysis with monoclonal antibodies and estimation of neurovirulence by mutant analysis by PCR and restriction enzyme cleavage (MAPREC) were performed for 13 type 1 strains of these isolates. The isolates were classified into three groups. Group I (five strains) had a variant type of antigenicity and neurovirulent phenotype. Group II (four strains) had the vaccine type of antigenicity and neurovirulent phenotype. Group III (four strains) had the vaccine type of antigenicity and an attenuated phenotype. Furthermore, it was demonstrated that the virulent isolates were neutralized by human sera obtained after oral poliomyelitis vaccine (OPV) administration, and the sera of rats immunized with inactivated poliovirus vaccine. Although vaccination was effective against virulent polioviruses, virulent viruses will continue to exist in the environment as long as OPV is in use.     

Characterization of CHAT and Cox type 1 live-attenuated poliovirus vaccine strains

CHAT and Cox type 1 live-attenuated poliovirus strains were developed in the 1950s to be used as vaccines for humans. This paper describes their characterization with respect to virulence, sensitivity for growth at high temperatures, and complete nucleotide and amino acid sequences. The results are compared to those for their common parental wild virus, the Mahoney strain, and to those for two other poliovirus strains derived from Mahoney, the Sabin 1 vaccine strain and the mouse-adapted LS-a virus. Analysis of four isolates from cases of vaccine-associated paralytic poliomyelitis related to the CHAT vaccine revealed genetic and phenotypic properties of the CHAT strain following replication in the human gut. CHAT-VAPP strain 134 contained a genome highly evolved from that of CHAT (1.1% nucleotide differences), suggesting long-term circulation of a vaccine-derived strain in the human population. The molecular mechanisms of attenuation and evolution of poliovirus in humans are discussed in the context of the global polio eradication initiative.     

A vision of a world without polio: the OPV cessation strategy.

Once the eradication of wild poliovirus has been confirmed, the public health benefits of routine immunization with OPV will no longer outweigh the burden of disease either due to paralysis caused by OPV (vaccine associated paralytic polio), or by outbreaks caused by circulating vaccine-derived polioviruses. The eventual cessation of OPV use in routine immunization programmes worldwide will become necessary to assure a lasting eradication of polio. As the world moves towards polio eradication and its certification, preparations are therefore being intensified for OPV cessation, and the risk management framework for safe OPV cessation is being put in place. The framework includes bio-containment of all known poliovirus and potentially infected substances, development of an international stockpile of oral polio vaccine, ensuring a mechanism for continued global surveillance and response for polio after eradication has been certified, and national policies if countries decide to continue vaccinating with inactivated polio vaccine (IPV). It is ironic that the vaccine on which the world has depended for polio eradication will itself become a risk to eradication once the transmission of wild poliovirus has been interrupted. Final preparations for the eventual global and simultaneous cessation of OPV will require the same level of international cooperation and coordination that has brought the world to the verge of polio eradication.     

Long-term circulation of vaccine-derived poliovirus that causes paralytic disease

Successful implementation of the global poliomyelitis eradication program raises the problem of vaccination against poliomyelitis in the posteradication era. One of the options under consideration envisions completely stopping worldwide the use of the Sabin vaccine. This strategy is based on the assumption that the natural circulation of attenuated strains and their derivatives is strictly limited. Here, we report the characterization of a highly evolved derivative of the Sabin vaccine strain isolated in a case of paralytic poliomyelitis from a 7-month-old immunocompetent baby in an apparently adequately immunized population. Analysis of the genome of this isolate showed that it is a double (type 1-type 2-type 1) vaccine-derived recombinant. The number of mutations accumulated in both the type 1-derived and type 2-derived portions of the recombinant genome suggests that both had diverged from their vaccine predecessors approximately 2 years before the onset of the illness. This fact, along with other recent observations, points to the possibility of long-term circulation of Sabin vaccine strain derivatives associated with an increase in their neurovirulence. Comparison of genomic sequences of this and other evolved vaccine-derived isolates reveals some general features of natural poliovirus evolution. They include a very high preponderance and nonrandom distribution of synonymous substitutions, conservation of secondary structures of important cis-acting elements of the genome, and an apparently adaptive character of most of the amino acid mutations, with only a few of them occurring in the antigenic determinants. Another interesting feature is a frequent occurrence of tripartite intertypic recombinants with either type 1 or type 3 homotypic genomic ends.     

Long-term excretion of vaccine-derived poliovirus by a healthy child

A child was found to be excreting type 1 vaccine-derived poliovirus (VDPV) with a 1.1% sequence drift from Sabin type 1 vaccine strain in the VP1 coding region 6 months after he was immunized with oral live polio vaccine. Seventeen type 1 poliovirus isolates were recovered from stools taken from this child during the following 4 months. Contrary to expectation, the child was not deficient in humoral immunity and showed high levels of serum neutralization against poliovirus. Selected virus isolates were characterized in terms of their antigenic properties, virulence in transgenic mice, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 1 strain. The VDPV isolates showed mutations at key nucleotide positions that correlated with the observed reversion to biological properties typical of wild polioviruses. A number of capsid mutations mapped at known antigenic sites leading to changes in the viral antigenic structure. Estimates of sequence evolution based on the accumulation of nucleotide changes in the VP1 coding region detected a "defective" molecular clock running at an apparent faster speed of 2.05% nucleotide changes per year versus 1% shown in previous studies. Remarkably, when compared to several type 1 VDPV strains of different origins, isolates from this child showed a much higher proportion of nonsynonymous versus synonymous nucleotide changes in the capsid coding region. This anomaly could explain the high VP1 sequence drift found and the ability of these virus strains to replicate in the gut for a longer period than expected.     

Structural and antigenic variation of the structural protein VP3 in serotype 1 poliovirus isolated from vaccinees

High resolution two-dimensional PAGE was used to analyse protein variation among serotype 1 poliovirus isolates. Viruses isolated from patients with recent histories of vaccination with live attenuated poliovirus were compared with prototype serotype 1 poliovaccine. The nonvaccine Mahoney and Brunenders strains of serotype 1 poliovirus were also analysed. The overall protein profile was conserved but the structural protein VP3 varied in its net charge among the viruses. Eight out of 14 clinical virus isolates had VP3 with a net basic charge identical to serotype 1 polio vaccine, whereas the remaining clinical isolates had an acidic VP3 similar to the nonvaccine type 1 strains. The altered VP3 mobility correlated with a change in antigenicity as determined by monoclonal antibodies directed to the neutralization site located on VP3. The data clearly illustrated the suitability of two-dimensional PAGE in analysing protein mutations in attenuated vaccine virus excreted by vaccinees.     

Environmental Poliovirus Surveillance during Oral Poliovirus Vaccine and Inactivated Poliovirus Vaccine Use in Córdoba Province,Argentina

This study compares the presence of environmental poliovirus in two Argentinean populations using oral poliovirus vaccine (OPV) or inactivated poliovirus vaccine (IPV). From January 2003 to December 2005, Córdoba City used IPV in routine infant immunizations, with the exception of intermittent OPV use in August 2005. Between May 2005 and April 2006, we collected weekly wastewater samples in Córdoba City and the province''s three major towns, which continued OPV use at all times. Wastewater samples were processed and analyzed for the presence of poliovirus according to WHO guidelines. During the months of IPV use in Córdoba City, the overall proportion of poliovirus-positive samples was 19%. During an intermittent switch from IPV to OPV, this proportion increased to 100% within 2 months. During the 3 months when IPV was reintroduced to replace OPV, a substantial proportion of samples (25%) remained positive for poliovirus. In the OPV-using sites, on average, 54% of samples were poliovirus positive. Seventy-seven percent of poliovirus isolates showed at least one mutation in the VP1-encoding sequence; the maximum genetic divergence from the Sabin strain was 0.7%. Several isolates showed mutations on attenuation markers in the VP1-encoding sequence. The frequency or type of virus mutation did not differ between periods of IPV and OPV use or by virus serotypes. This study indicates that the sustained transmission of OPV viruses was limited during IPV use in a middle-income country with a temperate climate. The continued importation of poliovirus and genetic instability of vaccine strains even in the absence of sustained circulation suggest that high poliovirus vaccine coverage has to be maintained for all countries until the risk of reintroduction of either wild or vaccine-derived poliovirus is close to zero worldwide.In the context of the near achievement of poliomyelitis eradication and anticipated cessation of oral poliovirus (PV) vaccine (OPV), the World Health Organization (WHO) has recommended the use of inactivated PV vaccine (IPV) in countries that have IPV production facilities or other countries where immunization programs fulfill certain financial and logistic criteria (37). IPV has been shown to be safe and immunogenic in children in both developed and developing countries.(34) IPV diminishes the excretion of PV by children challenged with the Sabin strain of PV only moderately. The questions of whether and to which extent Sabin PV that is reintroduced into a population immunized with IPV could establish circulation, mutate to vaccine-derived PV (VDPV), and consequently cause poliomyelitis remain important. No such emergence of VDPV in developed countries using IPV has been reported. However, suboptimal hygienic conditions and insufficient vaccine coverage in middle- or low-income countries could favor the establishment of PV circulation after reintroduction, as indicated by recent VDPV outbreaks in populations with low OPV coverage (27, 38).Argentina currently uses OPV in the childhood immunization program according to recommendations from the Pan-American Health Organization. The last case of poliomyelitis due to wild-type PV was reported in Argentina in 1984 and in Córdoba Province in 1971 (24). In Córdoba City, the capital of Córdoba Province, standalone IPV (Imovax Polio; Sanofi Pasteur) replaced OPV (Polioral; Novartis Vaccines) in the routine childhood immunization program (2, 4, and 6 months of age plus a booster at 18 months age) from 1 January 2003 to 31 December 2005, while the surrounding provinces continued to use OPV. Due to an IPV shortage between 10 August and 7 September 2005, OPV was used in the capital during this period. We conducted environmental PV surveillance in Córdoba Province from May 2005 to April 2006 to describe environmental PV circulation and molecular characteristics of PV depending on the vaccine used. In the present evaluation, we also describe the dynamic of PV circulation around the change of IPV-OPV-IPV-OPV in the capital. This observation can contribute evidence regarding the dynamics of PV circulation and its implication for global immunization policy after polio eradication.     

Intratypic recombination among lineages of type 1 vaccine-derived poliovirus emerging during chronic infection of an immunodeficient patient

We determined the complete genomic sequences of nine type 1 immunodeficient vaccine-derived poliovirus (iVDPV) isolates obtained over a 337-day period from a poliomyelitis patient from Taiwan with common variable immunodeficiency. The iVDPV isolates differed from the Sabin type 1 oral poliovirus vaccine (OPV) strain at 1.84% to 3.15% of total open reading frame positions and had diverged into at least five distinct lineages. Phylogenetic analysis suggested that the chronic infection was initiated by the fifth and last OPV dose, given 567 days before onset of paralysis, and that divergence of major lineages began very early in the chronic infection. Key determinants of attenuation in Sabin 1 had reverted in the iVDPV isolates, and representative isolates of each lineage showed increased neurovirulence for PVR-Tg21 transgenic mice. None of the isolates had retained the temperature-sensitive phenotype of Sabin 1. All isolates were antigenic variants of Sabin 1, having multiple amino acid substitutions within or near neutralizing antigenic sites 1, 2, and 3a. Antigenic divergence of the iVDPV variants from Sabin 1 followed two major independent evolutionary pathways. The emergence of distinct coreplicating lineages suggests that iVDPVs can replicate for many months at separate sites in the gastrointestinal tract. Some isolates had mosaic genome structures indicative of recombination across and within lineages. iVDPV excretion apparently ceased after 30 to 35 months of chronic infection. The appearance of a chronic VDPV excretor in a tropical, developing country has important implications for the strategy to stop OPV immunization after eradication of wild polioviruses.     

Changes in population dynamics during long-term evolution of sabin type 1 poliovirus in an immunodeficient patient

The evolution of the Sabin strain of type 1 poliovirus in a hypogammaglobulinemia patient for a period of 649 days is described. Twelve poliovirus isolates from sequential stool samples encompassing days 21 to 649 after vaccination with Sabin 1 were characterized in terms of their antigenic properties, virulence in transgenic mice, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin 1 strain. Poliovirus isolates from the immunodeficient patient evolved gradually toward non-temperature-sensitive and neurovirulent phenotypes, accumulating mutations at key nucleotide positions that correlated with the observed reversion to biological properties typical of wild polioviruses. Analysis of plaque-purified viruses from stool samples revealed complex genetic and evolutionary relationships between the poliovirus strains. The generation of various coevolving genetic lineages incorporating different mutations was observed at early stages of virus excretion. The main driving force for genetic diversity appeared to be the selection of mutations at attenuation sites, particularly in the 5' noncoding region and the VP1 BC loop. Recombination between virus strains from the two main lineages was observed between days 63 and 88. Genetic heterogeneity among plaque-purified viruses at each time point seemed to decrease with time, and only viruses belonging to a unique genotypic lineage were seen from day 105 after vaccination. The relevance of vaccine-derived poliovirus strains for disease surveillance and future polio immunization policies is discussed in the context of the Global Polio Eradication Initiative.     

Impact of exogenous sequences on the characteristics of an epidemic type 2 recombinant vaccine-derived poliovirus

Pathogenic circulating vaccine-derived polioviruses (cVDPVs) have become a major obstacle to the successful completion of the global polio eradication program. Most cVDPVs are recombinant between the oral poliovirus vaccine (OPV) and human enterovirus species C (HEV-C). To study the role of HEV-C sequences in the phenotype of cVDPVs, we generated a series of recombinants between a Madagascar cVDPV isolate and its parental OPV type 2 strain. Results indicated that the HEV-C sequences present in this cVDPV contribute to its characteristics, including pathogenicity, suggesting that interspecific recombination contributes to the phenotypic biodiversity of polioviruses and may favor the emergence of cVDPVs.     

Isolation and Characterization of a Type 2 Vaccine-Derived Poliovirus from Environmental Surveillance in China, 2012

Environmental surveillance of poliovirus on sewage has been conducted in Shandong Province, China since 2008. A type 2 vaccine-derived poliovirus (VDPV) with 7 mutations in VP1 coding region was isolated from the sewage collected in the city of Jinan in December 2012. The complete genome sequencing analysis of this isolate revealed 25 nucleotide substitutions, 7 of which resulted in amino acid alteration. No evidence of recombination with other poliovirus serotypes was observed. The virus did not lose temperature sensitive phenotype at 40°C. An estimation based on the evolution rate of the P1 coding region suggested that evolution time of this strain might be 160–176 days. VP1 sequence analysis revealed that this VDPV strain is of no close relationship with other local type 2 polioviruses (n = 66) from sewage collected between May 2012 and June 2013, suggesting the lack of its circulation in the local population. The person who excreted the virus was not known and no closely related virus was isolated in local population via acute flaccid paralysis surveillance. By far this is the first report of VDPV isolated from sewage in China, and these results underscore the value of environmental surveillance in the polio surveillance system even in countries with high rates of OPV coverage.     

Paralytic poliomyelitis in Russian Federation in 1998-2005

From 1998 through 2005 3,294 cases of acute flaccid paralysis (AFP) including 93 cases with clinical picture of poliomyelitis were registered in Russian Federation. From the latter cases 91 were classified as vaccine-associated paralytic poliomyelitis (VAPP): 66 were VAPP cases in oral poliomyelitis vaccine (OPV) recipients and 25--VAPP cases in contacts. VAPP rate was 1 case per 1.6 million of distributed OPV doses, 1 case per 2.2 million doses for OPV recipients, and 1 case per 186,000 doses for recipients of 1st OPV dose in children aged < 1 year. Majority of VAPP cases in recipients occurred after 1st dose (89.4%) and in contacts--in non-vaccinated children (76%). Mean interval between OPV administration and onset of VAPP in recipients was 21 days. Children aged < 1 year were predominant among VAPP cases (92.4% among recipient VAPP cases, and 80% among contact VAPP cases). Majority of the patients had unfavorable health status including defects of immunity. Most of poliovirus strains isolated from VAPP cases belonged to type 3 (52.9%) whereas to type 2 and 1--29.8% and 17.4% of strains respectively. All VAPP cases were associated with vaccine-derived polioviruses. A highly diverged poliovirus type 1 (2.65% of nucleotide substitutions in VP1 region) was isolated from patient with contact VAPP. Formation of poliovirus-neutralizing serum antibodies in children with VAPP including persons with immunodeficiency reflects the ability of the organism to produce specific antiviral immune response.     

Characterization of a highly evolved vaccine-derived poliovirus type 3 isolated from sewage in Estonia

Two types of vaccine-derived polioviruses have been recently designated to emphasize the different origins of the evolved viruses: circulating vaccine-derived polioviruses (cVDPV) associated with outbreaks of paralytic disease and strains isolated from chronically infected immunodeficient individuals (iVDPV). We describe here a type 3 VDPV (PV3/EST/02/E252; later E252) isolated from sewage collected in Tallinn, Estonia, in October 2002. Due to aberrant properties in subtyping, the virus was subjected to detailed characterization. Partial genomic sequencing suggested that the closest relative was the oral vaccine strain PV3/Sabin, but the two virus strains shared only 86.7% of the 900 nucleotides (nt) coding for the capsid protein VP1. Phylogenetic analysis of the nearly complete genome [nt 19 to poly(A)] revealed multiple nucleotide substitutions throughout the genome and a possible Sabin 3/Sabin 1-recombination junction site in the 2C coding region. A calculation based on the estimated mutation frequency of the P1 region of polioviruses suggested that the E252 virus might have replicated in one or more individuals for approximately 10 years. No persons chronically excreting poliovirus are known in Estonia. Amino acid substitutions were seen in all known antigenic sites, which was consistent with the observed aberrant antigenic properties of the virus demonstrated by both monoclonal antibodies and human sera from vaccinated children. In spite of the apparent transmission potential, no evidence was obtained for circulation of the virus in the Estonian population.     

Natural genetic exchanges between vaccine and wild poliovirus strains in humans

In a previous study of poliovirus vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis (VAPP) (9, 11), we reported that a high proportion (over 50%) of viruses had a recombinant genome. Most were intertypic vaccine/vaccine recombinants. However, some had restriction fragment length polymorphism (RFLP) profiles different from those of poliovirus vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin. To identify the parental wild donor of these sequences, we used RFLP profiles and nucleotide sequencing to look for similarity in the 3D polymerase-coding region of 61 wild, cocirculating poliovirus isolates (43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other four vaccine/wild recombinants, the wild parent could not be identified. The possibility that the wild sequences were of a non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an isolated phenomenon. We also found wild polioviruses (2 of 15) carrying vaccine-derived sequences in the 3' moiety of their genome. All these results suggest that genetic exchanges with wild poliovirus and perhaps with nonpoliovirus enteroviruses, are also a natural means of evolution for poliovirus vaccine strains.