Sensitivity of the acute flaccid paralysis surveillance system for poliovirus in South Africa, 2016-2019.

Introduction. Global poliovirus eradication is a public health emergency of international concern. The acute flaccid paralysis (AFP) surveillance programme in South Africa has been instrumental in eliminating polioviruses and keeping the country poliovirus free.Gap statement. The sensitivity of surveillance for polioviruses by every African country is of global interest in the effort to ensure global health security from poliovirus re-emergence.Aim. To describe the epidemiology of polioviruses from AFP cases and environmental samples in South Africa and to report the performance of the AFP surveillance system for the years 2016-2019 against targets established by the World Health Organization (WHO).Methods. Stool specimens from AFP or suspected AFP cases were received and tested as per WHO guidelines. Environmental samples were gathered from sites across the Gauteng province using the grab collection method. Concentration was effected by the two-phase polyethylene glycol method approved by the WHO. Suspected polioviruses were isolated in RD and/or L20B cell cultures through identification of typical cytopathic effects. The presence of polioviruses was confirmed by intratypic differentiation PCR. All polioviruses were sequenced using the Sanger method, and their VP1 gene analysed for mutations.Results. Data from 4597 samples (2385 cases) were analysed from the years 2016-2019. Two cases of immunodeficiency-associated vaccine-derived poliovirus (iVDPV) type 3 were detected in 2017 and 2018. A further 24 Sabin type 1 or type 3 polioviruses were detected for the 4 years. The national surveillance programme detected an average of 3.1 cases of AFP/100 000 individuals under 15 years old (2.8/100 000-3.5/100 000). The stool adequacy of the samples received was 53.0 % (47.0-55.0%), well below the WHO target of 80 % adequacy. More than 90 % of results were released from the laboratory within the turnaround time (96.6 %) and non-polio enteroviruses were detected in 11.6 % of all samples. Environmental surveillance detected non-polio enterovirus in 87.5 % of sewage samples and Sabin polioviruses in 12.5 % of samples.Conclusion. The AFP surveillance programme in South Africa is sensitive to detect polioviruses in South Africa and provided no evidence of wild poliovirus or VDPV circulation in the country.

Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game.

UNLABELLED: Vaccine manufacturing costs prevent a significant portion of the world's population from accessing protection from vaccine-preventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNA interference (RNAi) screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild-type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers >20-fold and >50-fold, respectively. Host gene knockdown events did not affect virus antigenicity, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced replication of enterovirus 71, a clinically relevant virus to which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing possibilities and should facilitate polio eradication using the inactivated poliovirus vaccine. IMPORTANCE: Using a genome-wide RNAi screen, a collection of host virus resistance genes was identified that, upon silencing, increased poliovirus and enterovirus 71 production by from 10-fold to >50-fold in a Vero vaccine manufacturing cell line. This report provides novel insights into enterovirus-host interactions and describes an approach to developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The results show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin strain) and wild-type polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine as well as further reduce costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine-preventable diseases.

[Complete Genome Characterization of Vaccine-derived Polioviruses Isolated in Yunnan,China].

To explore the genomic characterization of 4vaccine-derived poliovirus(VDPV)strains isolated from 2acute flaccid paralysis(AFP)cases in Yunnan Province in 2010 and 2012,respectively,the complete genome sequences of the 4strains were determined. Sequence analysis revealed that the complete genome length of the type Ⅱ and type Ⅰ VDPV was 7439nt and 7441 nt, respectively. Nucleotide and amino acid sequence similarities of type II VDPV were 95.4% and 97.7%,respectively,and type I VDPV were93.9% and 97.9%,respectively as compared with those of Sabin strains. Nucleotide substitutions were found at two important attenuation sites (nt 481 and nt in type Ⅱ VDPV, and three important attenuation sites(nt480,nt2795 and nt6203)in type I VDPV. Type 2 and type 1VDPV strains had 1.0% and2.3% divergence with Sabin strains, respectively. Similarity plot analysis showed multiple recombination events in the genome of the 4strains,which showed that the recombination was common and complex. Analysis of the characteristics of VDPVs on molecular level could provide valuable information on evolutionary dynamics and lay foundation for developing scientific and feasible strategy to control VDPV.

Highly divergent type 2 and 3 vaccine-derived polioviruses isolated from sewage in Tallinn, Estonia.

Highly divergent vaccine-derived polioviruses (VDPVs) have been isolated from sewage in Tallinn, Estonia, since 2002. Sequence analysis of VDPVs of serotypes 2 and 3 showed that they shared common noncapsid region recombination sites, indicating origination from a single trivalent oral polio vaccine dose, estimated to have been given between 1986 and 1998. The sewage isolates closely resemble VDPVs chronically excreted by persons with common variable immunodeficiency, but no chronic excretors have yet been identified in Estonia.

Poliovirus separation from cell extracts using capillary electrophoresis: potential use in vaccine production and control?

Rapid assessment of the concentration of virus particles in a given sample remains a challenge. Modern separation methods, such as capillary electrophoresis, were proposed recently to study viruses and viral infection or to separate and characterize viral vaccines in a time-efficient manner. Even though capillary electrophoresis is much more rapid than traditional virological methods and has the advantages of automation, increased precision and reliability, it has the drawback of reduced sensitivity for low concentrations. A sensitivity improvement is then necessary in many cases for a successful application. However, to date, only highly purified viral samples were examined using capillary electrophoresis. The injection of larger sample volumes, followed by intra-capillary concentration, was used in this study for cell extracts. Poliovirus was successfully detected rapidly, without any laborious staining procedures and incubation times. The method is simple, fast, automatic, requires only minute amounts of samples and reagents, and no expensive dyes or biological reagents. Additionally, the method showed a potential for monitoring the viral load during growth and purification, with obvious prospects for the optimization of the variable and time-consuming virus propagation procedures. The results of this study provide a potential basis for the development of routine methods for viral particles analysis, irrespective of their infective properties. In the future, the capillary electrophoresis test could help study the relationship between the intact poliovirus particles and the D-antigenic properties of a viral suspension, or could represent a supplementary or alternative test for virus concentration and D-antigen assays during vaccine production.

Phenotypic and genomic analysis of serotype 3 Sabin poliovirus vaccine produced in MRC-5 cell substrate.

The cell substrate has a pivotal role in live virus vaccines production. It is necessary to evaluate the effects of the cell substrate on the properties of the propagated viruses, especially in the case of viruses which are unstable genetically such as polioviruses, by monitoring the molecular and phenotypical characteristics of harvested viruses. To investigate the presence/absence of mutation(s), the near full-length genomic sequence of different harvests of the type 3 Sabin strain of poliovirus propagated in MRC-5 cells were determined. The sequences were compared with genomic sequences of different virus seeds, vaccines, and OPV-like isolates. Nearly complete genomic sequencing results, however, revealed no detectable mutations throughout the genome RNA-plaque purified (RSO)-derived monopool of type 3 OPVs manufactured in MRC-5. Thirty-six years of experience in OPV production, trend analysis, and vaccine surveillance also suggest that: (i) different monopools of serotype 3 OPV produced in MRC-5 retained their phenotypic characteristics (temperature sensitivity and neuroattenuation), (ii) MRC-5 cells support the production of acceptable virus yields, (iii) OPV replicated in the MRC-5 cell substrate is a highly efficient and safe vaccine. These results confirm previous reports that MRC-5 is a desirable cell substrate for the production of OPV.

Highly divergent neurovirulent vaccine-derived polioviruses of all three serotypes are recurrently detected in Finnish sewage.

In Finland, surveillance of potential re-emergence of poliovirus transmission is mainly based on environmental surveillance, i.e. search for infectious poliovirus in sewage samples. Since December 2008, 21 genetically highly divergent, neurovirulent vaccine-derived polioviruses (VDPV) have been isolated from sewage in Tampere, Finland. While the source of the VDPV is unknown, characteristics of the viruses resemble those of strains isolated from immunodeficient, persistently infected persons. No cases of suspected poliomyelitis have been reported in Finland since 1985.

[Analysis on identification and genetic character of type I vaccine-derived poliovirus in Shanxi province in 2007].

OBJECTIVE: To describe the source of vaccine-derived poliovirus (VDPV) and the effect on local polio-free status, the VP1 coding region was sequenced and analyzed for type I VDPV in Shanxi province in 2007. METHOD: The virus isolation was performed to double stool specimens from one case acute flaccid paralysis (AFP) patient. VP1 coding region of the isolated stain was sequenced and analyzed. The phylogenetic tree was constructed based on VP1 region sequence between Shanxi strains and other type I VDPVs. RESULT: 2 type I + II +III strains were isolated from double stool specimens from the AFP patient in Shanxi Province in 2007. VP1 sequencing of the two stains revealed > 1.0% divergence from the VP1 region of P I /Sabin vaccine strain. According to WHO criteria, the two stains were identified as type I vaccine-derived poliovirus (VDPV). Phylogenetic analysis based on VP1 coding sequence showed that the evolution distance of Shanxi type I VDPV was far away from other VDPVs detected in China. Moreover, no evidence supported the AFP patient as immunodeficiency patient. So Shanxi type I VDPVs were classified into ambiguous VDPV(aVDPV). CONCLUSION: Considering the genetic character for Shanxi type I VDPV and the local OPV coverage, we highly suspected that an immunodeficiency patient in local area who long-term excreted VDPVs existed and resulted in the patient infection of VDPV in Shanxi in 2007. In the post era of polio eradication, the detection and management for the possible existing patient of long-term excretion VDPV should be strengthened.

Reemergence of recombinant vaccine-derived poliovirus outbreak in Madagascar.

BACKGROUND: After the 2001-2002 poliomyelitis outbreak due to recombinant vaccine-derived polioviruses (VDPVs) in the Toliara province of Madagascar, another outbreak reoccurred in the same province in 2005. METHODS: We conducted epidemiological and virological investigations for each polio case patient and for their contacts. RESULTS: From May to August 2005, a total of 5 cases of acute flaccid paralysis were reported among unvaccinated or partially vaccinated children 2-3 years old. Type-3 or type-2 VDPV was isolated from case patients and from healthy contacts. These strains were classified into 4 recombinant lineages that showed complex mosaic genomic structures originating from different vaccine strain serotypes and probably from human enterovirus C (HEV-C) species. Genetic relatedness could be observed among these 4 lineages. Vaccination coverage of the population was very low (<50%). CONCLUSIONS: The broad distribution of VDPVs in the province and their close genetic relationship indicate intense and rapid cocirculation and coevolution of the vaccine strains and of their related HEV-C strains. The occurrence of an outbreak due to VDPV 3 years after a previous outbreak indicates that a short period with low vaccination coverage is enough to create favorable conditions for the emergence of VDPV in this setting.

Circulación silenciosa de poliovirus demostrada por interferencia a los enterovirus no polio / Silent circulation of poliovirus demonstrated by interference against non-polio enterovirus

Se obtuvieron muestras de heces semanalmente, en niños menores de 3 años para aislar los poliovirus y enterovirus no polio, con el objetivo de incrementar el conocimiento de las circulaciones de los derivados de la vacuna durante las campañas masivas. Los esquemas de vacunación continua permiten la circulación de esos virus durante grandes períodos de tiempo. En los niños se demostró una interferencia de los enterovirus no polio por los poliovirus vacunales. Sin embargo, mientras los bajos porcentajes de enterovirus no polio no mostraron diferencias significativas, sí se encontró en los altos porcentajes de poliovirus vacunales aislados en niños menores de 1 año en relación con los de 1 y 2 años. Basados en esa contradicción, se estimó la circulación silenciosa de los poliovirus por cálculos matemáticos. Con los poliovirus estimados se permitió obtener las curvas simuladas. Posteriormente, en otra investigación se confirmaron los resultados por métodos inmunológicos(AU)

Isolation and characterization of circulating type 1 vaccine-derived poliovirus from sewage and stream waters in Hispaniola.

Twenty-one cases of acute flaccid paralysis (AFP) were reported on the island of Hispaniola in 2000. Laboratory analysis confirmed the presence of circulating vaccine-derived poliovirus (cVDPV) type 1 in stool samples obtained from patients. As a complement to the active search for cases of AFP, environmental sampling was conducted during November and December 2000, to test for cVDPV in sewage, streams, canals, and public latrines. Fifty-five environmental samples were obtained and analyzed for the presence of polioviruses by use of cell culture followed by neutralization and reverse-transcription polymerase chain reaction. Of the 23 positive samples, 10 tested positive for poliovirus type 1, 7 tested positive for poliovirus type 2, 5 tested positive for poliovirus type 3, and 1 tested positive for both poliovirus type 2 and type 3. By sequence analysis of the complete viral capsid gene 1 (VP1), a 2.1%-3.7% genetic sequence difference between 7 type 1 strains and Sabin type 1 vaccine strain was found. Phylogenetic analysis showed that these viruses are highly related to cVDPV isolated from clinical cases and form distinct subclusters related to geographic region. Our findings demonstrate a useful role for environmental surveillance of neurovirulent polioviruses in the overall polio eradication program.

Detection and growth kinetics of SV40 preparations with different enhancer element copy number.

It has been reported that the rate of growth of SV40 in certain cell culture systems is affected by the number of copies of the enhancer element, and that this might in turn affect the ability of manufacturers of polio vaccines to detect SV40 contamination in tests for adventitious agents. Eleven strains of SV40 of which three were primary isolates, three were control strains used in different laboratories and five were well characterised were examined for their enhancer copy number. It was found that the three primary isolates contained a single copy while three preparations used as controls in different laboratories were mixtures of single and multiple copy strains. The remaining five strains consisted of one known double enhancer and four known single enhancer strains. All eleven strains were titrated in BSC-1 cells. There was no correlation between the number of copies of the enhancer element and the rate of growth or the final titre reached, although there was variation between the strains.