Poliovirus Strains Research Articles

A New Live-Attenuated Poliovirus Vaccine Constructed by Rational Design Improves Safety by Preventing Reversion to Virulence

The life-attenuated Sabin oral poliovirus vaccine (OPV), created over 60 years ago, replicate efficiently in the vaccinee’s gut and, therefore, elicits robust mucosal and systemic immunity, which limits poliovirus spread. Accordingly, OPV has been used in the context of the Global Polio Eradication Initiative, which aims to end the circulation of all polioviruses. However, the program has met a significant hurdle imposed by the ability of OPV to evolve to regain transmissibility and virulence. This represents a risk of widespread polio re-emergence should immunization cease. We exploited molecular features of poliovirus replication to develop a safer, genetically stable, strain by limiting its ability to increase fitness and regain neurovirulence. This novel attenuated poliovirus strain is immunogenic in preclinical and clinical tests and may enable completion of poliovirus eradication as well as pave the way for the rational design of effective and safe vaccines for polio-related pathogens.

Use of hyperattenuated poliovirus as a replacement for Sabin or wild-type strains for laboratory assays in a post eradication world

Poliovirus serotype 2 (PV2) has been declared eradicated and was removed from the live vaccine programme in 2016. WHO have developed the Global Action Plan (GAPIII) to describe containment conditions for laboratory work with poliovirus after eradication, and in the UK, all PV2 has been reclassified to hazard group 3 (HG3). Laboratory work involving live poliovirus will continue to be required years after eradication for the purposes of: vaccine testing; environmental surveillance; immunoglobulin testing; and for the development of new public health interventions. Compliance with GAPIII and HG3 containment conditions would impose financial burden and failure would lead to disruption to essential activities. Development of safer poliovirus strains for use outside of containment could overcome these issues. The S19 poliovirus strain, designed to be hyperattenuated and extremely genetically stable, was used as a cassette for the introduction of capsid proteins of other strains and serotypes (Knowlson et al. 2015), originally for vaccine production. S19 viruses are unable to infect transgenic mice (by intraspinal inoculation) or non-human primates (by mouth) at very high doses and are unlikely to infect humans at biological temperature. PV2 S19 strains have recently been approved for use outside of GAPIII containment requirements by the Containment Advisory Group. Validation of strains for laboratory assays and a highly sensitive QC assay based on NGS will be described.

Differences in Antigenic Structure of Inactivated Polio Vaccines Made From Sabin Live-Attenuated and Wild-Type Poliovirus Strains: Impact on Vaccine Potency Assays.

Following the declaration of wild-type 2 poliovirus eradication in 2015, the type 2 component was removed from the live-attenuated oral polio vaccine (OPV). This change implies a need to improve global coverage through routine immunization with inactivated polio vaccine (IPV), to ensure type 2 immunity. Several manufacturers use Sabin OPV strains for IPV production (sIPV), rather than the usual wild-type strains used for conventional IPV (cIPV). However, in contrast to cIPV, potency assays for sIPV have not been standardized, no international references exist, and no antigen units have been defined for a sIPV human dose. Thus, sIPV products from different manufacturers cannot be compared, and the relationship between antigenicity and immunogenicity of sIPV is not well understood. A collaborative study was conducted in which laboratories used different methods to measure the antigen content of a set of sIPV and cIPV samples with an aim to identify a suitable reference for sIPV products. The study revealed differences in the reactivity of antibody reagents to cIPV and sIPV products. Homologous references are required to measure the antigen content of IPV products consistently. The first World Health Organization international standard for sIPV was established, with new, specific Sabin D-antigen units assigned.

Continued use of poliovirus after eradication: hyper-attenuated strains as a safe alternative for release testing of human immunoglobulins.

Wild-type poliovirus may be eradicated soon and under WHO GAPIII guidance, laboratory use will be discontinued or subject to strict containment. Per US Code of Federal Regulations, however, immunoglobulin lot release testing will still require use of replicating poliovirus. The suitability of S19 hyper-attenuated and apathogenic poliovirus strains as alternatives to the currently used wild-type virus in such a release assay was investigated. S19 poliovirus strains were propagated in a commercial setting using good virological practices and maintenance of the S19 hyper-attenuated genotype was confirmed by massively parallel sequencing. The attenuated phenotype of the produced S19 stocks was confirmed in a highly sensitive mouse-model. Equivalency in performance was seen in the lot release assay for the S19 and wild-type polioviruses. The deployment of such hyper-attenuated and thoroughly characterized S19 stocks in these and other essential activities might reconcile final containment measures with continued safe use of poliovirus.

Poliovirus Detection and Genetic Characteristic from Sewage in Heilongjiang Province from 2013 to 2016.

By monitoring the sewage system in Heilongjiang province from 2013 to 2016, this study aimed to analyze the epidemiological tendency and genetic mutation of poliovirus (PV) found in the environment in order to setup a warning system for vaccine-derived poliovirus (VDPV) and the spread of wild poliovirus. In this study, we collected 139 sewage samples from 8 regions in Heilongjiang province. Poliovirus was identified from 72 samples, and the positivity rate was 51%. A total of 263 PV strains were isolated, including 22 strains of type 1 PV, 104 strains of type 2 PV, and 137 strains of type 3 PV. As a result of intratypic differentiation, using real-time PCR and nucleotide sequencing, 3 type 1 pre-VDPV, one type 2 VDPV, and 2 type 3 pre-VDPV strains were isolated. Interestingly, one type 1 strain with 5 nucleotide deletions and one type 3 recombinant on VP1 were isolated. By continuously monitoring the poliovirus in the environment, we aimed to recognize the VDPV or wild poliovirus with high neurovirulence from large-scale circulation and set up a warning system to avoid morbidity and virus transmission.

Poliomyelitis in modern conditions: achievements and prospects

The creation in the middle of the 20th century vaccines against poliomyelitis (PM) – inactivated vaccine (IPV) and live oral vaccine from Sabin strains (OPV) with various properties, advantages and disadvantages, but highly effective, made it possible to implement the idea of elimination of PM. Since 1988, the WHO Global Program of PM eradication has achieved remarkable success: the incidence of PM caused by wild poliovirus (PV) has been reduced by 10 thousand times, the number of endemic countries has been reduced to 3, the circulation of wild PV has been discontinued in 4 regions of the world the wild type 2 of PV has been eradicated, and wild type 3 of PV has not been detected for almost 5 years. Under conditions of a decrease in the incidence of PM caused by wild PV, the known negative properties of trivalent OPV made its further use problematic. These negative properties are: 1) the ability to cause post-vaccination complications and 2) the genetic instability of Sabin strains, especially PV of type 2, and their ability under certain conditions (primarily in conditions of low collective immunity to PV) to quickly restore neurovirulence, transforming into circulating vaccinederived PV (VDPV), capable of causing incidents and outbreaks of PM. In order to reduce the risk associated primarily with type 2 PV, WHO proposed a global switch to the use of bivalent OPV from types 1 and 3, completed in 2016. In 2019, WHO plans to complete eradication of type 1 and 3 PVs, and in 2022 completely abandon the OPV. The precondition for the safety of such tactics is the maintenance of high collective immunity to PM. There are several threats to the security of this strategy. PVs can “silently” circulate in the human population for a long time without clinical manifestations of PM, which, with inadequate epidemiological surveillance can lead to the return of PM. The reintroduction of both wild PV and Sabin strains can occur from institutions that preserve / work with PV. The source of VDPV can be people with primary immunodeficiencies, which continuously excrete PV. It is necessary to maintain surveillance over the PM, expand additional types of surveillance for the PV, strict containment of all PVs. The only way to maintain collective immunity is immunization with trivalent IPV. The current global shortage of IPV poses a significant threat to the world’s epidemiological well-being. The solution to the problem is the development of a new generation of safe and effective vaccines, improving the ways of introducing IPV, developing antiviral drugs.

Quantitative multiplex one-step RT-PCR assay for identification and quantitation of Sabin strains of poliovirus in clinical and environmental specimens

An improved quantitative multiplex one-step RT-PCR (qmosRT-PCR) for simultaneous identification and quantitation of all three serotypes of poliovirus is described. It is based on using serotype-specific primers and fluorescent TaqMan oligonucleotide probes. The assay can be used for high-throughput screening of samples for the presence of poliovirus, poliovirus surveillance and for evaluation of virus shedding by vaccine recipients in clinical trials to assess mucosal immunity. It could replace conventional methods based on cell culture virus isolation followed by serotyping. The assay takes only few hours, and was found to be simple, specific, sensitive and has large quantitative linearity range. In addition, the method could be used as readout in PCR-based poliovirus titration and neutralization assays.

Isolation of Sabin-like Polioviruses from Sewage in Poland.

As a complement to the active search for cases of acute flaccid paralysis, environmental sampling was conducted from January to December 2011, to test for any putative polio revertants and recombinants in sewage. A total of 165 environmental samples were obtained and analyzed for the presence of polioviruses by use of cell culture (L20B, RD and Caco-2) followed by neutralization and reverse-transcription polymerase chain reaction. Out of the 31 CPE positive samples, 26 contained one and 5 two different serotypes, yielding a total of 36 PVs. The microneutralization test revealed the presence of 7, 10 and 19 strains belonging to poliovirus serotype 1, 2 and 3, respectively. The genomic variability of 36 poliovirus strains was examined by the restriction fragment length polymorphism assay (RFLP). By combined analyses of two distant, polymorphic segments of the viral genome, one situated in the capsid protein VP1 coding region and the other in the 3D-polymerase coding region, we screened for the putative poliovirus revertants and recombinants. All detected PVs were classified as vaccine strains on the basis of RFLP-VP1 test. None of wild-type PVs or vaccine derived polioviruses were detected. RFLP assay also revealed the presence of 11 recombinants in 3D-polymerase coding region. Nine isolates appeared to be S3/S2, one S3/S1 and S1/S2 recombinant in analyzed 3Dpol region. This study revealed, through environmental monitoring, the introduction of SL PVs into the population associated with the routine use of OPV in Poland before the April 2016. Our findings demonstrate the usefulness of environmental surveillance in the overall polio eradication program.

Successes and challenges of expansion of environmental poliovirus surveillance in the WHO South-East Asia Region.

The last decade has witnessed an exponential expansion of environmental surveillance (ES) of poliovirus in sewage samples in the World Health Organization (WHO) South-East Asia Region. This has grown from only three sites in Mumbai, India in 2001 to 56 sites in 2017 in Bangladesh, India, Indonesia, Myanmar, Nepal and Thailand. ES is critical to the region in providing evidence of silent transmission of vaccine-derived poliovirus and Sabin-like poliovirus type 2 - especially since the global "switch" to cease use of oral polio vaccine type 2 - and for monitoring the effectiveness of containment activities. This targeted expansion of ES to supplement surveillance for acute flaccid paralysis (AFP) required quality assurance in ES procedures, improvements in the sensitivity of the laboratory-based surveillance system, and establishment of real-time data analysis for evidence-based programmes. ES in the region has provided documentary evidence for the absence of indigenous wild poliovirus in circulation and no importations via international travellers. Post-switch, while no vaccine-derived poliovirus was detected from AFP cases, ES identified five ambiguous vaccine-derived polioviruses in 2016 and early 2017, with no evidence of circulation. Future challenges include monitoring for vaccine-derived poliovirus strains shed for a prolonged time by immunodeficient individuals, and expanding ES to areas lacking sewage networks. To maintain the polio-free status of the WHO South-East Asia Region and achieve a world free of poliomyelitis, critical evaluation of immunization coverage, continued performance of surveillance for acute flaccid paralysis, and enhanced analysis of sewage samples to detect any breach in containment are essential.

Isolation of Vaccine-Like Poliovirus Strains in Sewage Samples From the United Kingdom.

Environmental surveillance (ES) is a sensitive method for detecting human enterovirus (HEV) circulation, and it is used worldwide to support global polio eradication. We describe a novel ES approach using next-generation sequencing (NGS) to identify HEVs in sewage samples collected in London, United Kingdom, from June 2016 to May 2017. Two different methods were used to process raw sewage specimens: a 2-phase aqueous separation system and size exclusion by filtration and centrifugation. HEVs were isolated using cell cultures and analyzed using NGS. Type 1 and 3 vaccine-like poliovirus (PV) strains were detected in samples collected from September 2016 through January 2017. NGS analysis allowed us to rapidly obtain whole-genome sequences of PV and non-PV HEV strains. As many as 6 virus strains from different HEV serotypes were identified in a single cell culture flask. PV isolates contained only a small number of mutations from vaccine strains commonly seen in early isolates from vaccinees. Our ES setup has high sensitivity for polio and non-PV HEV detection, generating nearly whole-genome sequence information. Such ES systems provide critical information to assist the polio eradication endgame and contribute to the improvement of our understanding of HEV circulation patterns in humans.

Etiologic Involvement of Enterovirus and Human Bocavirus in Acute Flaccid Paralysis Cases in India

BackgroundAcute flaccid paralysis (AFP), characterized by the rapid onset of asymmetric paralysis, can be caused by a variety of viral infections or coinfections. Besides wild-type and revertant vaccine strains of polioviruses, several nonpolio enteroviruses, have also been associated with AFP. Enteroviruses (EVs) are RNA viruses in the family Picornaviridae comprising more than 100 serotypes that are divided into four species, human enteroviruses A to D. The clinical manifestations of EVs range from conjunctivitis, respiratory tract infection, myocarditis, meningitis, encephalitis, and neonatal sepsis, like illness. Human Bocavirus (HBoV), a newly classified member of the Parvoviridae family, has been detected frequently in feces of diarrhoeic children suggesting its possible etiological involvement in the disease.MethodsTotal 586 stool specimens were collected in 2016 from children suspected for AFP. Molecular method for targeting 5’ untranslated region (UTR) and VP1 capsid region was used for detection of human enteroviruses (HEV), human boca viruses (HBoV) and saffold viruses in direct clinical specimen.ResultsEV RNA was detected in 103 (17.6%) of 586 stool specimens by real-time RT-PCR targeting the highly conserved 5’ UTR region. Out of them, 71 (12.11%) were NPEV, partially sequenced by VP1 which revealed the prevalence of echovirus (ECV) 19 (n = 6), ECV 11 (n = 7), ECV 18 (n = 4), ECV 33 (n = 5), ECV 29 (n = 1), ECV 25 (n = 2), ECV 24 (n = 3), ECV 3 (n = 3), ECV 14 (n = 2), ECV 13 (n = 1), ECV 2 (n = 1), ECV 20 (n = 2), ECV 27 (n = 4), ECV 6 (n = 2), CV A10 (n = 2), CV A9 (n = 1), CV A6 (n = 2), CV B4 (n = 1), CV B5 (n = 3), CV B6 (n = 3), EV 80 (n = 1), EV 83 (n = 1), EV 97 (n = 2).Total 63 (10.75%) HBoVs were detected by real-time PCR which were further sequenced by VP1, consists of HBoV-1 (n = 8), HBoV-2 (n = 15), HBoV-3 (n = 9) and HBoV-4 (n = 5). Out of them 9 (1.5%) were detected as co infection with NPEVs. Phylogenetic analysis showed 0.9 - 5.6% divergence at nucleotide level among HBoVs.Total 9 (1.5%) saffold viruses was detected and characterized by VP1 sequencing.ConclusionECV and HBoV were found the main etiologic agent in children suspected with AFP. Molecular typing of these viruses is useful for characterizing emerging serotypes and their epidemiological investigation.Disclosures All authors: No reported disclosures.

Live attenuated enterovirus vaccine (OPV) is not associated with islet autoimmunity in children with genetic susceptibility to type 1 diabetes: prospective cohort study.

Animal and human studies have implied that enterovirus infections may modulate the risk of islet autoimmunity and type 1 diabetes. We set out to assess whether serial administration of live oral poliovirus vaccine (OPV) in early life can influence the initiation of islet autoimmunity in a cohort of genetically predisposed children. OPV was administered to 64 children and a further 251 children received inactivated poliovirus vaccine (IPV). The emergence of type 1 diabetes-associated autoantibodies in serum (autoantibodies to GAD, insulinoma-associated protein 2, insulin and islet cells) was monitored during prospective follow-up. Stool and serum samples were collected for enterovirus detection by RT-PCR. Administration of OPV increased enterovirus detected in stool samples from 11.3% to 38.9% (p<0.001) during the first year of life. During the follow-up (median 11.0years), at least one autoantibody was detected in 17.2% of children vaccinated with OPV and 19.1% with IPV (p=0.723). At least two autoantibodies were observed in 3.1% and 6.8% of children, respectively (p=0.384). Replication of attenuated poliovirus strains in gut mucosa is not associated with an increased risk of islet autoimmunity. ClinicalTrials.gov : NCT02961595.

Polio and Measles Down the Drain: Environmental Enterovirus Surveillance in the Netherlands, 2005 to 2015.

Polioviruses (PVs) are members of the genus Enterovirus In the Netherlands, the exclusion of PV circulation is based on clinical enterovirus (EV) surveillance (CEVS) of EV-positive cases and routine environmental EV surveillance (EEVS) conducted on sewage samples collected in the region of the Netherlands where vaccination coverage is low due to religious reasons. We compared the EEVS data to those of the CEVS to gain insight into the relevance of EEVS for poliovirus and nonpolio enterovirus surveillance. Following the polio outbreak in Syria, EEVS was performed at the primary refugee center in Ter Apel in the Netherlands, and data were compared to those of CEVS and EEVS. Furthermore, we assessed the feasibility of poliovirus detection by EEVS using measles virus detection in sewage during a measles outbreak as a proxy. Two Sabin-like PVs were found in routine EEVS, 11 Sabin-like PVs were detected in the CEVS, and one Sabin-like PV was found in the Ter Apel sewage. We observed significant differences between the three programs regarding which EVs were found. In 6 sewage samples collected during the measles outbreak in 2013, measles virus RNA was detected in regions where measles cases were identified. In conclusion, we detected PVs, nonpolio EVs, and measles virus in sewage and showed that environmental surveillance is useful for poliovirus detection in the Netherlands, where live oral poliovirus vaccine is not used and communities with lower vaccination coverage exist. EEVS led to the detection of EV types not seen in the CEVS, showing that EEVS is complementary to CEVS.IMPORTANCE We show that environmental enterovirus surveillance complements clinical enterovirus surveillance for poliovirus detection, or exclusion, and for nonpolio enterovirus surveillance. Even in the presence of adequate surveillance, only a very limited number of Sabin-like poliovirus strains were detected in a 10-year period, and no signs of transmission of oral polio vaccine (OPV) strains were found in a country using exclusively inactivated polio vaccine (IPV). Measles viruses can be detected during an outbreak in sewage samples collected and concentrated following procedures used for environmental enterovirus surveillance.

Patients with Primary Immunodeficiencies Are a Reservoir of Poliovirus and a Risk to Polio Eradication.

Immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) have been isolated from primary immunodeficiency (PID) patients exposed to oral poliovirus vaccine (OPV). Patients may excrete poliovirus strains for months or years; the excreted viruses are frequently highly divergent from the parental OPV and have been shown to be as neurovirulent as wild virus. Thus, these patients represent a potential reservoir for transmission of neurovirulent polioviruses in the post-eradication era. In support of WHO recommendations to better estimate the prevalence of poliovirus excreters among PIDs and characterize genetic evolution of these strains, 635 patients including 570 with primary antibody deficiencies and 65 combined immunodeficiencies were studied from 13 OPV-using countries. Two stool samples were collected over 4 days, tested for enterovirus, and the poliovirus positive samples were sequenced. Thirteen patients (2%) excreted polioviruses, most for less than 2 months following identification of infection. Five (0.8%) were classified as iVDPVs (only in combined immunodeficiencies and mostly poliovirus serotype 2). Non-polio enteroviruses were detected in 30 patients (4.7%). Patients with combined immunodeficiencies had increased risk of delayed poliovirus clearance compared to primary antibody deficiencies. Usually, iVDPV was detected in subjects with combined immunodeficiencies in a short period of time after OPV exposure, most for less than 6 months. Surveillance for poliovirus excretion among PID patients should be reinforced until polio eradication is certified and the use of OPV is stopped. Survival rates among PID patients are improving in lower and middle income countries, and iVDPV excreters are identified more frequently. Antivirals or enhanced immunotherapies presently in development represent the only potential means to manage the treatment of prolonged excreters and the risk they present to the polio endgame.

Isolation and characterization of a highly evolved type 3 vaccine-derived poliovirus in China

In this study, we report the identification and characterization of a highly evolved type 3 vaccine-derived poliovirus (VDPV) strain designated as WIV14, isolated in 2014 from a 4-year-old child suspected of having an enteroviral infection in China. Complete genome sequence of WIV14 revealed multiple nucleotide substitutions when compared with the attenuated poliovirus (PV) Sabin 3, including the reversion of three major attenuation sites to wild type. From the nucleotide divergence for the P1/capsid region, we estimated that the evolution time of WIV14 was more than 7 years, indicating the possible long time of replication. WIV14 strain seemed to have differences in biological characteristics compared with attenuated PV strains, such as being non-temperature-sensitive and producing large plaques. The current isolation of a highly divergent type 3 VDPV gives an idea of the risk of emergent VDPV strains, and emphasizes the importance of maintaining high vaccination coverage and herd immunity against PVs in China.

Scientific consultation on the safety and containment of new poliovirus strains for vaccine production, clinical/regulatory testing and research. Report of a meeting held at NIBSC, Potters Bar, Hertfordshire, UK, 6/7th July 2016

When poliomyelitis is totally eradicated from the natural world containment will be vital to prevent its re-emergence. The matter has become pressing as type 2 component of oral polio vaccine was completely withdrawn by May 2016 as wild ty[e 2 was declared eradicated. Work on polioviruses must be contained in accordance with GAPIII (the third version of the Global Action Plan of WHO). Some activities will be essential for years after eradication. Vaccine production and control, surveillance and supportive applied and academic research must all continue. Most laboratories do not currently comply with GAPIII and could not do so in the short term without disruption of essential activities including vaccine supply. The development and use of safer strains is raised in GAPIII and the meeting considered the strains available and the uses to which they could be put to facilitate compliance with the aims of GAPIII.

Global polio eradication: Where are we in Europe and what next?

The world was never so close to reach the polio eradication: only 37 cases notified in 2016 in only three countries, but the game is not yet at the end. The risk of polio outbreaks in the EU is smaller than it has ever been in the past, but it is not so small that we can ignore it. The EU MS must remain alert and plan and prepare for managing polio events or outbreaks because of the possible dire consequences. The IPV only vaccination schedule universally applied in EU has achieved satisfactory coverage, but constantly leaving small accumulating pockets of susceptible individuals. Moreover the IPV only schedule is not an absolute barrier against poliovirus silent transmission as demonstrated in the recent Israel outbreak. The availability of annually revised S.O.P. from WHO GPEI on the identification and response of a polio event, without local poliovirus transmission or a polio outbreak with sustained transmission, helps and challenge EU countries to update their polio national preparedness plans. The EU/EEA area, in fact, is a peculiar area regarding the polio risk both for its vaccination policy, the large polio vaccines manufactures and the constant immigration from areas at polio high risk, but also EU include cultural and financial potentials crucial to sustain the polio end game strategy and reach the benefit of a world without polio risk. Poliovirus eradication will continue to be challenged as long as there is the worldwide presence of polioviruses in laboratories and vaccine production plants. Most of the world’s OPV vaccines are produced in the EU and many laboratories and research centers store and handle polio viruses. EU Member States are engaged actively in implementing the poliovirus biocontainment plans that are part of the polio eradication strategy and to certify the destruction of poliovirus strains and potentially contaminated biological materials.

Characterization of human monoclonal antibodies that neutralize multiple poliovirus serotypes.

Following the eradication of wild poliovirus (PV), achieving and maintaining a polio-free status will require eliminating potentially pathogenic PV strains derived from the oral attenuated vaccine. For this purpose, a combination of non-cross-resistant drugs, such as small molecules and neutralizing monoclonal antibodies (mAbs), may be ideal. We previously isolated chimpanzee and human mAbs capable of neutralizing multiple PV types (cross-neutralization). Here, we describe three additional human mAbs that neutralize types 1 and 2 PV and one mAb that neutralizes all three types. Most bind conformational epitopes and have unusually long heavy chain complementarity determining 3 domains (HC CDR3). We assessed the ability of the mAbs to neutralize A12 escape mutant PV strains, and found that the neutralizing activities of the mAbs were disrupted by different amino acid substitutions. Competitive binding studies further suggested that the specific mAb:PV interactions that enable cross-neutralization differ among mAbs and serotypes. All of the cloned mAbs bind PV in the vicinity of the "canyon", a circular depression around the 5-fold axis of symmetry through which PV recognizes its cellular receptor. We were unable to generate escape mutants to two of the mAbs, suggesting that their epitopes are important for the PV life cycle. These data indicate that PV cross-neutralization involves binding to highly conserved structures within the canyon that binds to the cellular receptor. These may be facilitated by the long HC CDR3 domains, which may adopt alternative binding configurations. We propose that the human and chimpanzee mAbs described here could have potential as anti-PV therapeutics.

Genotype distribution of human enteroviruses isolated from swage in Shanghai during year 2013-2014

Objective: To explore the time and genotype distribution of human enterovirus (HEV) isolated from sewage in Shanghai in 2013-2014. Methods: One sewage sample each was collected from two local sewage plants located in Minhang District and Jiading District on the same day at the day 24-28 of every month from 2013 to 2014. Each sample weighed 1 L. The specimens were concentrated by anionic membrane absorption, eluted with beef extract solution, and then used to inoculate RD, HEp-2, and L20B cell lines. A total of 249 enterovirus strains were isolated from sewage samples during the study period, including 185 non-polio enterovirus (NPEV) and 64 poliovirus (PV) strains, which were identified as vaccine strains. RT-PCR and Sanger sequencing were performed to identify HEV genotypes. Homologous analysis of VP1 sequences was conducted using BioEdit (version 7.0.0). Phylogenetic analysis was performed using the neighbor-joining method based on the alignment of VP1 gene sequences using MEGA (version 4.0.2). Results: Among 185 NPEV strains, 178 strains were successfully sequenced and classified into 15 genotypes, including coxsackievirus group B (CVB) 2, 3, and 5; enteric cytopathic human orphan (ECHO) virus 1, 3, 6, 7, 11, 13, 19, 20, 24, 25, and 30; and coxsackievirus group A 4. CVB5 and ECHO6 genotypes accounted for 33.5% (56 strains) and 24.9% (43 strains) of NPEV isolates, respectively. During the study period, HEV isolates were mainly isolated in summer and autumn in Minhang District. ECHO6 strains were frequently isolated from June 2013 to July 2014. Thereafter, the number of ECHO6 strains gradually reduced in the second half of 2014. CVB5 strains demonstrated scattered distribution from 2013 to the first half of 2014 and gradually increased in the second half of 2014. The distribution of ECHO6 and CVB5 strains in Jiading District was similar to that in Minhang District. In 2013-2014, CVB5 strains comprised C6 and C8 subgenotypes, which belong to two transmission chains and show large differences compared with foreign strains isolated during the same period. ECHO6 strains comprised C6, C8, and D9 subgenotypes, which belong to three transmission chains. Moreover, ECHO6 subgenotype D9 was a dominant subgenotype in Shanghai, with broad geographical distribution both at home and abroad. Conclusion: Poliovirus was identified as a vaccine strain in environmental surveillance from June 2013 to April 2014 in Shanghai. Several transmission strains of ECHO6 and CVB5 were identified, which were the dominant serotypes.

Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication.

The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals.In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated.Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field.