Spread of vaccine-derived poliovirus from a paralytic case in an immunodeficient child: an insight into the natural evolution of oral polio vaccine.

Sabin strains used in the manufacture of oral polio vaccine (OPV) replicate in the human organism and can give rise to vaccine-derived polioviruses. The increased neurovirulence of vaccine derivatives has been known since the beginning of OPV use, but their ability to establish circulation in communities has been recognized only recently during the latest stages of the polio eradication campaign. This important observation called for studies of their emergence and evolution as well as extensive surveillance to determine the scope of this phenomenon. Here, we present the results of a study of vaccine-derived isolates from an immunocompromised poliomyelitis patient, the contacts, and the local sewage. All isolates were identified as closely related and slightly evolved vaccine derivatives with a recombinant type 2/type 1 genome. The strains also shared several amino acid substitutions including a mutation in the VP1 protein that was previously shown to be associated with the loss of attenuation. Another mutation in the VP3 protein resulted in altered immunological properties of the isolates, possibly facilitating virus spread in immunized populations. The patterns and rates of the accumulation of synonymous mutations in isolates collected from the patient over the extended period of excretion suggest either a substantially nonuniform rate of mutagenesis throughout the genome, or, more likely, the strains may have been intratypic recombinants between coevolving derivatives with different degrees of divergence from the vaccine parent. This study provides insight into the early stages of the establishment of circulation by runaway vaccine strains.

Poliovirus-binding inhibition ELISA for evaluation of immune response to oral poliovirus vaccine: a possible alternative to the neutralization test.

This study describes an ELISA variant (Binding Inhibition ELISA, BI ELISA) for the quantitative determination of neutralization-relevant antibodies to polioviruses. The test differs from previously described analogs by utilizing polyclonal immune reagents: capture antibodies and biotin-labeled detecting antibodies. The BI ELISA was compared with the conventional neutralization test (NT) by testing live Sabin and wild-type poliovirus strains. The comparison of 68 serum samples taken from Oral Poliovirus Vaccine (OPV) recipients showed 100% specificity and sensitivity for Sabin 1 and Sabin 2, and 100% sensitivity and 97% specificity for Sabin 3. Good correlations (r = 0.7, 0.77, 0.65 for Sabin 1, 2, 3, respectively) were demonstrated between the NT and BI ELISA results. These results indicate that the BI ELISA can be used as a reliable alternative to the NT for determination of neutralization-relevant antibodies to polioviruses in vaccinees and in large-scale sero-epidemiological studies.

Reevaluation of nucleotide sequences of wild-type and attenuated polioviruses of type 3.

Published sequences of wild-type and attenuated Sabin strains of type 3 poliovirus (Leon/37 and Leon 12a(1)b) were derived from cDNA clones. Recent direct sequencing of Sabin 3 RNA showed that it differed from the published sequence in at least two sites. Here results of direct sequencing of genomes of three independently re-derived sub-strains of attenuated Sabin 3 poliovirus used for oral poliovirus vaccine (OPV) production in addition to the most widely used Pfizer sub-strain are reported. The results showed that all four sub-strains of attenuated type 3 poliovirus contain unique patterns of mutations. Two stocks of the wild-type progenitor Leon/37 strain were also sequenced. Analysis of the two samples of Leon/37 virus showed that one of them is much closer to the Sabin 3 strain, and is an intermediate product of the attenuation process. In addition, we created genetically engineered constructs which contained some of the mutations suspected for their possible role in neurovirulence, and tested them in monkeys and in transgenic mice sensitive to poliovirus. The results suggested that none of them increased neurovirulence of the virus, but some may improve virus replication. Therefore the only mutation occurring in Sabin 3 under vaccine production conditions that appears to affect neurovirulence of the virus is the well known U-->C reversion at nucleotide 472.

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.

Genetic stability of Sabin 1 strain of poliovirus: implications for quality control of oral poliovirus vaccine.

The Sabin vaccine strains of poliovirus, like all RNA viruses, exist as a quasispecies of genomic sequences whose composition can be altered during virus propagation. Since changes in vaccine virus during manufacture can enhance the neurovirulent potential of the vaccine, each monovalent lot of oral poliovirus vaccine (OPV) undergoes several tests to ensure consistency of manufacture, including the monkey neurovirulence test (MNVT). Recently, we proposed a new molecular approach for direct quantification of vaccine variants with neurovirulent potential as an alternative way to monitor consistency of OPV production. Analysis of the Sabin 1 genome allowed us to identify a limited number of specific loci that exhibit significant change during viral propagation in vitro and in vivo. Here we explore the possible roles of these changes and show that 7427-U-->C and 7441-G-->A alterations in the 3'-UTR of the Sabin 1 virus do not increase monkey neurovirulence. These, as well as our previous results, suggest that only mutations in the 5'-UTR play a significant role in the limited increase in Sabin 1 monkey neurovirulence observed after extended propagation of the virus beyond the passage level used in vaccine production. Our studies with high-passage batches of the Sabin 1 strain confirmed the stability of this strain, which retains acceptable levels of monkey neurovirulence even after serial passages at elevated temperature. Compared to the MNVT, molecular analysis of the genetic composition of Sabin 1 poliovirus provides a more sensitive analytical approach to monitor consistency of vaccine production.

Limited genetic changes in the Sabin 1 strain of poliovirus occurring in the central nervous system of monkeys.

Replication of attenuated poliovirus strains results in their partial deattenuation. Recently we identified mutations accumulating in the Sabin 1 poliovirus in cell cultures. Here we report genetic changes occurring in this virus during replication in the central nervous system (CNS) of monkeys. Viruses isolated from different parts of the CNS of rhesus monkeys (inoculated into the spinal cord) were screened for sequence heterogeneities and newly identified mutations were independently confirmed and quantified using mutant analysis by PCR and restriction enzyme cleavage (MAPREC). All consistently accumulating mutations identified in this study were located in untranslated regions: GU-->AU or GU-->GC substitution at a complementary pair formed by nucleotides 480 and 525, U-->C substitution at nucleotide 612, and GU-->AU or GU-->GC substitution of a base pair formed by the nucleotides 7427/7441 immediately preceding the poly(A) tract. All these mutations except one (7427) were previously identified in cell culture passages or stool isolates from vaccinees. Sequencing of 11 CNS isolates also identified a few random silent mutations that accumulated as neutral 'passengers', passively co-selected with genuinely selectable mutations present on the same RNA molecule. One isolate also contained the wild-type base at nucleotide 2741 (Ala88-->Thr in VP1). Our results demonstrate a remarkable genetic stability of the Sabin 1 poliovirus in the CNS of monkeys, suggesting that deattenuation is determined by a very limited number of mutations. These mutations can be assayed by MAPREC to monitor the consistency of oral poliovirus vaccine (OPV) production.

Microevolution of type 3 Sabin strain of poliovirus in cell cultures and its implications for oral poliovirus vaccine quality control.

Screening for sequence heterogeneities in Sabin Type 3 strains of attenuated poliovirus demonstrated mutations that consistently accumulate to significant levels following 10 passages in cultures of primary African green monkey kidney (AGMK) cells or continuous cultures of Vero cells. Fourteen newly identified mutations were quantified by mutant analysis by PCR and restriction enzyme cleavage in passages and in batches of commercial vaccines made in AGMK and Vero cells from the Sabin original (SO) seed virus and from a seed virus rederived by RNA plaque purification (RSO or "Pfizer" seed). Nine of the 14 mutations were reproducibly observed in more than one series of passages. Although 5 other mutations were observed in only one set of passages each, their content gradually increased to a high percentage, suggesting that all the mutations that we found accumulated consistently. SO-derived samples accumulated more mutations than did RSO-derived ones, and the number of mutations and the rates of their accumulation were higher in Vero than in AGMK cells. While the rates of accumulation of most mutations were higher when passaging was performed at 37 degrees, a U-->C transition at nucleotide 5832 occurred faster at 34 degrees, the temperature used for vaccine production. Analysis of Type 3 oral poliovirus vaccine (OPV) monopools made by six manufacturers found only 5 of these newly identified mutations in vaccine batches (nucleotides 3956, 4935, 5357, 5788, and 5832). Some of the mutations were found in trace amounts (less than 0.1%) while others were present at up to 1.8% levels. The pattern of these mutations was characteristic for the type of seed virus and the cell substrate but demonstrated no correlation with results of the monkey neurovirulence test. Therefore the only mutation occurring in Type 3 OPV which contributed to neurovirulence in monkeys was the previously described reversion at nucleotide 472. Quantitation of reversion at nucleotide 472 can be utilized for assessment of acceptability of vaccine lots, while other mutations can be used for monitoring the consistency of vaccine production.

Genetic stability and mutant selection in Sabin 2 strain of oral poliovirus vaccine grown under different cell culture conditions.

Mutations that consistently accumulated in the attenuated Sabin 2 strain of poliovirus during propagation in cell cultures were identified by sequence heterogeneity assay and quantified by mutant analysis by PCR and restriction enzyme cleavage (MAPREC). Eight additional sites previously identified in stool isolates were also examined by MAPREC in the virus passages. The pattern of selectable mutations and the rate of their accumulation depended on the type and confluence of the cell culture and the temperature of virus growth. Five unstable genomic sites were identified in Sabin 2 virus passaged 10 times at 34 degrees in African green monkey kidney (AGMK) cells, with the mutations accumulating in the range 1 to 24%. Accumulation of these mutations did not appear to result in a loss of attenuated phenotype since the virus passaged under these conditions passed the monkey neurovirulence test (MNVT). The content of the 481-G revertant known to be related to neurovirulence in monkeys did not increase. Thus, our results suggest that upon growth of Sabin 2 virus in AGMK cells at 34 degrees, the key determinant(s) of attenuation remained stable, and the mutations that occurred did not affect monkey neurovirulence. In virus passaged 10 times at 37 degrees in AGMK cells, 4 unstable genomic sites were identified, in some of them accumulating up to 12% of the mutants. This virus sample severely failed the MNVT. Virus passaged in Vero cells at 34 and 37 degrees accumulated mutants at 7 and 14 genomic sites, respectively, including 481-G in both cases, with almost complete substitution of the original nucleotides at some of the sites. We tested 44 commercial monopools of Type 2 OPV and found out that all of them contained 481-G revertants in the range 0.4-1.1%. An increase in the 481-G revertants in passaged viruses to the level of 4% and above correlated with failure of these samples by the MNVT. Since the pattern of selectable mutations differed in viruses grown in the two cell cultures used in this study, specific mutation profiles should be determined for each cell substrate used for vaccine production to assess manufacturing consistency.

Microevolution of Sabin 1 strain in vitro and genetic stability of oral poliovirus vaccine.

Mutants consistently accumulating in Sabin 1 poliovirus during serial passaging in vitro were identified by sequence heterogeneity assay and quantitated using mutant analysis by PCR and restriction enzyme cleavage (MAPREC). Only four unstable genomic sites were identified in virus passaged 10 times in African green monkey kidney (AGMK) cells, and eight sites in virus passaged in Vero cells. Mutations accumulated both in untranslated regions of RNA (nucleotides 480, 525 and 7441) and in coding sequences, as missense (nucleotides 1449, 4944, and 6203) or silent (nucleotides 1123 and 1141) mutations. The most prominent selectable mutations were found at complementary nucleotides 480 and 525 of the 5'-untranslated region (5'-UTR) of the Sabin strain, changing the G:U pair in F-domain to either A:U or G:C variants. These two variants have been shown previously to have an increased neurovirulence in monkeys. The G:C variant accumulated during passage in Vero cells, while A:U variant accumulated in CV-1 cells. Virus passaged in AGMK cells accumulated both variants. Higher temperature (37 instead of 34 degrees) strongly favored selection of mutants in Vero cells, had a smaller effect on mutant accumulation in AGMK cells, and had no effect in CV-1 cells. Monopools of type 1 oral poliovirus vaccine (OPV) made by seven manufacturers were found to contain both 480-A and 525-C revertants at a combined level of 1.1-2.7%. Viral samples with increased amounts of these revertants had higher neurovirulence in monkeys. Our results suggest that quantitation of these reversions by MAPREC may be prognostic for results of the monkey neurovirulence test (MNVT) and can be used for monitoring type 1 OPV consistency.

[Virus-specific proteins and RNA of the virus of hemorrhagic fever with renal syndrome]. / Virusspetsificheskie belki i RNK virusa gemorragicheskoi lokhoradki s pochechnym sindromom.

Virus-specific proteins G1, G2, and N with molecular weights of 70, 55-57, and 50 kilodaltons, respectively, were detected by radioimmunodiffusion tests in VERO E-6 cells infected with strains of virus of hemorrhagic fever with renal syndrome (HFRS) isolated in the European USSR from a patient with HFRS, a fatal human case of HFRS (the strains K-27 and P-360) and from a bank vole (strain CG-1820). The sera from human convalescents after HFRS in the European USSR and rat sera prepared with the CG-1820 strain precipitated proteins possessing similar electrophoretic characteristics from a lysate of cells infected with the CG-1820, K-27 and P-360 strains. The sera from human HFRS convalescents in the Far East did not precipitate protein Gl. The viral RNA derived by immunosorption method from intracellular nucleocapsids of CG-1820 strain and strain 4590 (isolated from Ap. Peninsulae in the Far East) contained 3 classes of molecules: L, M, and S. L- and M-RNA of these strains had the same molecular weight (1.62 and 1.38 megadaltons). The molecular weight of S-RNA of the strain 4590 was 0.76 megadalton and that of the CG-1820 strain 0.83 megadalton. It is assumed that there are species differences among the viruses, causative agents of HFRS, circulating in the European and Far East regions of the USSR.

[Area and natural reservoirs of hemorrhagic fever virus with kidney syndrome in the Far East of the USSR]. / Areal i prirodnye rezervuary virusa gemorragicheskoi likhoradki s pochechnym sindromom na dal'nem vostoke SSSR.

Lungs of 9127 small mammals of 17 species trapped in Khabarovsk region, Magadan, Amur, and Sakhalin regions in 1982-1987 were examined, among them 11 species are reservoirs of HFRS virus. Most frequently infected are striped field mice and Japanese field mice, red voles and large-toothed red-backed voles which are the dominant species of the appropriate landscape formations. Circulation of two HFRS virus serotypes among small mammals was demonstrated. The main epidemiological role belongs to the striped field mouse in HFRS foci of the meadow-field type, and to Asiatic field mouse in forest foci in the territories examined.

Detection of the antigen and antibodies to the eastern subtype of haemorrhagic fever with renal syndrome virus in small rodents in Slovakia.

Direct enzyme-linked immunosorbent assay (ELISA) was used for the demonstration of haemorrhagic fever with renal syndrome (HFRS) virus antigen in lung tissue of small rodents trapped in Eastern and Western Slovakia. The eastern subtype of HFRS virus antigen was demonstrated in the lungs of Apodemus agrarius and of the western subtype in the lungs of Microtus arvalis. Antibodies to HFRS virus antigen have been detected in Apodemus species (A. agrarius and A. flavicollis) in higher titres to the Eastern subtype.

Virus-specific proteins and RNAs from cells infected with hemorrhagic fever with renal syndrome (HFRS) viruses derived from endemic areas of the U.S.S.R.

Cell-associated proteins of rodent (CG-1820 strain) and human (K-27 and 360 strains) hantaviruses isolated in the European endemic areas of the U.S.S.R. are antigenically similar as revealed by immunoprecipitation and immunoblotting assays. Nucleocapsid-associated RNAs of representative hantaviruses of four antigenic groups [Sugiyama et al. (1987) J Gen Virol 68: 979-987] have unique PAGE patterns. "Electropherotyping" of the RNAs isolated from infected cells might be used for identifying and distinguishing hantaviruses.

[Circulation of the virus of hemorrhagic fever with renal syndrome among small mammals in the territory of the USSR]. / Izuchenie tsirkuliatsii virusa gemorragicheskoi likhoradki s pochechnym sindromom sredi melkikh mlekopitaiushchikh na territorii SSSR.

Over 55,000 small mammals of 72 species trapped in 62 administrative territories practically in all landscape-geographical zones of the USSR were examined in 1980-1985. The use of current laboratory methods demonstrated a wide spread of HFRS virus in the territory of this country, involvement in the epizootic process of most species of forest and steppe murine rodents and insectivora. In each landscape zone, the main carriers of the HFRS agent were established, represented, as a rule, by the basic species having a high and stable population density.

[Detection of natural foci of the hemorrhagic fever with renal syndrome in the Georgian SSR]. / Obnaruzhenie prirodnykh ochagov gemorragicheskoi likhoradki s pochechnym sindromom v Gruzinskoi SSR.

The circulation of the causative agent of hemorrhagic fever with renal syndrome (HFRS) in Georgia was studied. Altogether, 7,938 small mammals of 16 species trapped in 23 administrative areas of the republic were examined, and the specific antigen of HFRS virus was detected in 308 (3.8%). In serological examinations of 10 patients, antibody to HFRS virus was found in 4. Analysis of the above results indicates the existence of natural foci of HFRS in the Georgian SSR.

[Exact determination of the eastern border of the European portion of the nosogeographic range of the hemorrhagic fever with renal syndrome]. / Ob utochnenii vostochnoi granitsy evropeiskoi chasti nozoareala gemorragicheskoi likhoradki s pochechnym sindromom.

Radioimmunoassay was used for examinations of 21,488 serum specimens from the population of 145 areas of 8 regions and 2 republics. The immune portion comprised 8.8% to the west of the assumed border and 2.7% to the east (t = 20.7). Active natural foci of the infection were found in individual territories located to the east of the assumed border (areas of Saratov Province to the north of the Irgiz River, trans-Ural areas of Bashkiria, southern taiga areas of Sverdlovsk, Tyumen, and northwestern Omsk Provinces). The extreme eastern points of the endemic area are Tevriz, Tary, and Znamenskoe Districts of Omsk Province. The areas of Siberian forest steppe, the steppe areas of western Siberia and those on the left bank of the Volga (Zavolzhye) are free from the infection foci. Natural foci of infection were found in the taiga part of the Komi ASSR, whereas they were absent in the forest tundra zone.

[Detection of foci of hemorrhagic fever with renal syndrome in the Estonian SSR]. / Obnaruzhenie ochagov gemorragicheskoi likhoradki s pochechnym sindromom v Estonskoi SSR.

Examination by the enzyme-immunoassay of organs of 10 small mammal species trapped in different landscape zones of the Estonian SSR revealed the presence of HFRS virus antigen in organs of bank voles and field mice. Radioimmunoassay studies of serum specimens from donors demonstrated the presence of antibody to HFRS virus in 2.54% of those examined.

[Evaluation of the results of a direct solid-phase immunoenzyme method for determining antigens]. / Otsenka rezul'tatov priamogo tverdofaznogo immunofermentnogo metoda dlia opredeleniia antigenov.

Aspects of an objective analysis of the results of solid-phase enzyme-immunoassay (SPEIA) for identification of antigens (a direct version) were considered on the model of the virus of hemorrhagic fever with renal syndrome. Using a statistical method, certain parameters of 11 types of polystyrene and polyvinylchloride microplates (solid carrier) of different manufacturers were studied from the point of view of their suitability for obtaining reproducible SPEIA results. Statistical methods for the evaluation of EIA data are proposed to increase the effectiveness of the use of this test and making it quite an objective one.

[Time periods of antibody preservation in patients convalescing from hemorrhagic fever with renal syndrome in European foci of the infection]. / O srokakh sokhraneniia antitel u rekonvalestsentov gemorragicheskoi likhoradki s pochechnym sindromom v evropeiskikh ochagakh infektsii.

The radioimmunoassay of serum samples from 76 convalescents after hemorrhagic fever with the renal syndrome (HFRS), that took place in 1964 in Ufa, revealed the presence of specific antibodies in 75% of the convalescents. The absence of antibodies may be attributed both to their loss in some of the convalescents and to mistakes in the clinical diagnosis. The study of serum samples from 19 convalescents who had the infection in 1960 during the laboratory outbreak of HFRS at the Gamaleya Institute of Epidemiology and Microbiology (Moscow) showed the presence of antibodies in all convalescents. In both groups the infection was linked with common red-backed voles (the Western serological variant of the virus).

[Results of a humoral immunity study in hemorrhagic fever with renal syndrome in the Maritime Territory]. / Rezul'taty izucheniia gumoral'nogo immuniteta pri gemorragicheskoi likhoradke s pochechnym sindromom v Primorskom krae.

The paper presents the results of serological diagnosis of hemorrhagic fever with renal syndrome (HFRS) and studies of the time course of humoral immunity formation in this disease carried out in 1980-1983 in the Maritime Territory, one of the active HFRS foci.