[An oligonucleotide microarray for detection and discrimination of orthopoxviruses based on oligonucleotide sequences of two viral genes].

An oligonucleotide microarray for detection and identification of orthopoxviruses was developed. Genus specific and orthopoxvirus species-specific regions of the genes encoding chemokine binding and alpha/beta-interferon binding proteins were used as a target. The developed microarray allows the variola, monkeypox, cowpox, vaccinia, camel-pox and ectromelia (mousepox) viruses to be distinguished with a high degree of reliability.

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.

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 protein 3A inhibits tumor necrosis factor (TNF)-induced apoptosis by eliminating the TNF receptor from the cell surface.

Viral infections often trigger host defensive reactions by activating intrinsic (intracellular) and extrinsic (receptor-mediated) apoptotic pathways. Poliovirus is known to encode an antiapoptotic function(s) suppressing the intrinsic pathway. Here, the effect of poliovirus nonstructural proteins on cell sensitivity to tumor necrosis factor (TNF)-induced (i.e., receptor-mediated) apoptosis was studied. This sensitivity is dramatically enhanced by the viral proteinase 2A, due, most likely, to inhibition of cellular translation. On the other hand, cells expressing poliovirus noncapsid proteins 3A and 2B exhibit strong TNF resistance. Expression of 3A neutralizes the proapoptotic activity of 2A and results in a specific suppression of TNF signaling, including the lack of activation of NF-kappaB, due to elimination of the TNF receptor from the cell surface. In agreement with this, poliovirus infection results in a dramatic decrease in TNF receptor abundance on the surfaces of infected cells as early as 4 h postinfection. Poliovirus proteins that confer resistance to TNF interfere with endoplasmic reticulum-Golgi protein trafficking, and their effect on TNF signaling can be imitated by brefeldin A, suggesting that the mechanism of poliovirus-mediated resistance to TNF is a result of aberrant TNF receptor trafficking.

Increased safety level of serotype 3 Sabin oral poliomyelitis vaccine lots by improved seed virus, and tissue culture and virus infection conditions.

The content of 472U to 472C revertant virus in serotype 3 oral poliomyelitis monovalent bulk vaccines can be quantified by MAPREC (Mutant Analysis by PCR and Restriction Enzyme Cleavage). Besides other wildtype reversions identified in propagated type 3 Sabin strain populations, the 472U to 472C reversion correlates most prominently with neurovirulence in the monkey neurovirulence test. Therefore, the results can be used for the discrimination of 'good' and 'bad' vaccines on the molecular level. In international collaborative studies it has been well established that vaccine lots containing revertant genomes below a critical threshold pass the in vivo monkey neurovirulence test (MNVT), while vaccine lots containing more revertants fail the MNVT. In this communication we show that the MAPREC test is a sensitive tool for quality control and the demonstration of consistency in large scale production. Furthermore, MAPREC offers a possibility to assess the effect of changed production conditions on the rate of reversion and to find conditions for consistent production with low reversion rates.

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.

PCR engineering of viral quasispecies: a new method to preserve and manipulate genetic diversity of RNA virus populations.

A PCR-based method for the controlled manipulation of individual genomic sites of poliovirus with concomitant preservation of the sequence heterogeneity of the rest of the genome is proposed. The new approach can be used for the creation of stable DNA repositories of populations of extremely heterogenous RNA viruses and may have implications for live vaccine technology.

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 --> lle 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 degrees, it does not threaten the safety of OPV.

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.

Studies on neurovirulence in poliovirus-sensitive transgenic mice and cynomolgus monkeys for the different temperature-sensitive viruses derived from the Sabin type 3 virus.

We have studied methods for testing the neurovirulence of live poliovaccine viruses by intraspinal inoculation into mice carrying the human poliovirus receptor gene (Tg mice). A comparison of the neurovirulence of Sabin type 3 vaccine virus and related viruses using the 50% paralysis dose determined after intraspinal inoculation into the Tg mice as an index revealed a close correlation between the results of the paralysis dose in Tg mice, the neurovirulence expressed by the histopathological lesions core in monkeys, and the temperature sensitivity of the viruses. The results of experiments in the Tg mice also showed a good correlation with the number of mutations at position 472 from U to C in the 5' noncoding region in the genomes of the viruses tested. These results strongly suggest that the neurovirulence test for oral poliomyelitis vaccine using the Tg mice is an excellent method and may be used in place of the test using monkeys.

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.

Use of anti-TNF-alpha antiserum to investigate toxic alveolitis arising from cotton dust exposure.

Cotton dust has been associated with byssinosis and toxic alveolitis. A murine animal model has been developed with which to investigate the pathogenesis of these disorders. Studies with the model have reproduced the neutrophilic inflammation characteristic of the alveolitis, and have shown the presence of tumor necrosis factor-alpha (TNF-alpha) in the bronchoalveolar lavage (BAL) fluid. The current study investigated the role of TNF-alpha in the inflammatory response by use of a polyclonal antiserum to recombinant murine TNF-alpha. Following a 4-h exposure to cotton dust, experimental animals showed a 40-fold increase in BAL cells with 92% neutrophils. There was a 24-fold increase in TNF-alpha in the BAL fluid. Up regulation of TNF-alpha mRNA expression was detected in BAL cells. Mice pretreated with anti-TNA-alpha antiserum displayed a marked attenuation of the neutrophilic inflammation; however, the level of TNF-alpha mRNA expression was not reduced in these mice. These studies support a major role of TNF-alpha in the toxic alveolitis induced by cotton dust inhalation.

Consistent selection of mutations in the 5'-untranslated region of oral poliovirus vaccine upon passaging in vitro.

We have previously found that upon passaging type 3 oral poliovirus vaccine (OPV) in cell cultures the proportion of revertants at nucleotide 472 rapidly increases [Chumakov et al.: Proceedings of the National Academy of Sciences of the United States of America 88:199-203 1991]. Systematic study on the accumulation of these revertants showed that it was dependent on the multiplicity of infection and the temperature at which virus was grown. Revertants at position 472 of type 3 OPV accumulated faster in vaccines derived from Sabin Original (SO) substrain than from RNA-plaque purified (RSO) substrain. The rate of accumulation of 472-C revertants differed among cell lines and was higher in overgrown cell cultures suggesting that host factors are involved in the selection of mutants. We also found that accumulation of mutants occurred in vitro at position 480 in type 1 and position 481 in type 2 OPV, making the selection for revertants in domain F of the 5'-noncoding region a general phenomenon for all three Sabin strains. Assessment of the abundance of these mutants may be used for evaluation of the quality of OPV lots.

Quantitative aspects of the mutant analysis by PCR and restriction enzyme cleavage (MAPREC).

Quantitation of virus revertants by PCR and restriction enzyme cleavage may give nonlinear results and, in some cases, produce artifacts caused by nucleotide misincorporation and heteroduplex formation, occurring during PCR. Modifications of the procedure allowed us to overcome these problems and develop a highly sensitive and reliable method of mutant quantitation. This procedure can be used to assess the quality of live vaccines and to study heterogeneity of viral and bacterial populations.