Cara C. Burns

Modulation of poliovirus replicative fitness in hela cells by deoptimization of synonymous codon usage in the capsid region

ABSTRACT We replaced degenerate codons for nine amino acids within the capsid region of the Sabin type 2 oral poliovirus vaccine strain with corresponding nonpreferred synonymous codons. Codon replacements were introduced into four contiguous intervals spanning 97% of the capsid region. In the capsid region of the most highly modified virus construct, the effective number of codons used (NC) fell from 56.2 to 29.8, the number of CG dinucleotides rose from 97 to 302, and the G+C content increased from 48.4% to 56.4%. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased in proportion to the number of replacement codons. Plaque areas decreased over an ∼10-fold range, and virus yields decreased over an ∼65-fold range. Perhaps unexpectedly, the synthesis and processing of viral proteins appeared to be largely unaltered by the restriction in codon usage. In contrast, total yields of viral RNA in infected cells were reduced ∼3-fold and specific infectivities of purified virions (measured by particle/PFU ratios) decreased ∼18-fold in the most highly modified virus. The replicative fitness of both codon replacement viruses and unmodified viruses increased with the passage number in HeLa cells. After 25 serial passages (∼50 replication cycles), most codon replacements were retained, and the relative fitness of the modified viruses remained well below that of the unmodified virus. The increased replicative fitness of high-passage modified virus was associated with the elimination of several CG dinucleotides. Potential applications for the systematic modulation of poliovirus replicative fitness by deoptimization of codon usage are discussed.

Vaccine-Derived Polioviruses

The attenuated oral poliovirus vaccine (OPV) has many properties favoring its use in polio eradication: ease of administration, efficient induction of intestinal immunity, induction of durable humoral immunity, and low cost. Despite these advantages, OPV has the disadvantage of genetic instability, resulting in rare and sporadic cases of vaccine-associated paralytic poliomyelitis (VAPP) and the emergence of genetically divergent vaccine-derived polioviruses (VDPVs). Whereas VAPP is an adverse event following exposure to OPV, VDPVs are polioviruses whose genetic properties indicate prolonged replication or transmission. Three categories of VDPVs are recognized: (1) circulating VDPVs (cVDPVs) from outbreaks in settings of low OPV coverage, (2) immunodeficiency-associated VDPVs (iVDPVs) from individuals with primary immunodeficiencies, and (3) ambiguous VDPVs (aVDPVs), which cannot be definitively assigned to either of the first 2 categories. Because most VDPVs are type 2, the World Health Organizations plans call for coordinated worldwide replacement of trivalent OPV with bivalent OPV containing poliovirus types 1 and 3.

Genetic Inactivation of Poliovirus Infectivity by Increasing the Frequencies of CpG and UpA Dinucleotides within and across Synonymous Capsid Region Codons

ABSTRACT Replicative fitness of poliovirus can be modulated systematically by replacement of preferred capsid region codons with synonymous unpreferred codons. To determine the key genetic contributors to fitness reduction, we introduced different sets of synonymous codons into the capsid coding region of an infectious clone derived from the type 2 prototype strain MEF-1. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased sharply with increased frequencies of the dinucleotides CpG (suppressed in higher eukaryotes and most RNA viruses) and UpA (suppressed nearly universally). Replacement of MEF-1 capsid codons with the corresponding codons from another type 2 prototype strain (Lansing), a randomization of MEF-1 synonymous codons, increased the %G+C without increasing CpG, and reductions in the effective number of codons used had much smaller individual effects on fitness. Poliovirus fitness was reduced to the threshold of viability when CpG and UpA dinucleotides were saturated within and across synonymous codons of a capsid region interval representing only ∼9% of the total genome. Codon replacements were associated with moderate decreases in total virion production but large decreases in the specific infectivities of intact poliovirions and viral RNAs. Replication of codon replacement viruses, but not MEF-1, was temperature sensitive at 39.5°C. Synthesis and processing of viral intracellular proteins were largely unaltered in most codon replacement constructs. Replacement of natural codons with synonymous codons with increased frequencies of CpG and UpA dinucleotides may offer a general approach to the development of attenuated vaccines with well-defined antigenicities and very high genetic stabilities.

Outbreak of Type 2 Vaccine-Derived Poliovirus in Nigeria: Emergence and Widespread Circulation in an Underimmunized Population

Wild poliovirus has remained endemic in northern Nigeria because of low coverage achieved in the routine immunization program and in supplementary immunization activities (SIAs). An outbreak of infection involving 315 cases of type 2 circulating vaccine-derived poliovirus (cVDPV2; >1% divergent from Sabin 2) occurred during July 2005–June 2010, a period when 23 of 34 SIAs used monovalent or bivalent oral poliovirus vaccine (OPV) lacking Sabin 2. In addition, 21 “pre-VDPV2” (0.5%–1.0% divergent) cases occurred during this period. Both cVDPV and pre-VDPV cases were clinically indistinguishable from cases due to wild poliovirus. The monthly incidence of cases increased sharply in early 2009, as more children aged without trivalent OPV SIAs. Cumulative state incidence of pre-VDPV2/cVDPV2 was correlated with low childhood immunization against poliovirus type 2 assessed by various means. Strengthened routine immunization programs in countries with suboptimal coverage and balanced use of OPV formulations in SIAs are necessary to minimize risks of VDPV emergence and circulation.

Vaccine-Derived Poliomyelitis 12 Years after Infection in Minnesota

A 44-year-old woman with long-standing common variable immunodeficiency who was receiving intravenous immune globulin suddenly had paralysis of all four limbs and the respiratory muscles, resulting in death. Type 2 vaccine-derived poliovirus was isolated from stool. The viral capsid protein VP1 region had diverged from the vaccine strain at 12.3% of nucleotide positions, and the two attenuating substitutions had reverted to the wild-type sequence. Infection probably occurred 11.9 years earlier (95% confidence interval [CI], 10.9 to 13.2), when her child received the oral poliovirus vaccine. No secondary cases were identified among close contacts or 2038 screened health care workers. Patients with common variable immunodeficiency can be chronically infected with poliovirus, and poliomyelitis can develop despite treatment with intravenous immune globulin.

Molecular epidemiology of silent introduction and sustained transmission of wild poliovirus type 1, Israel, 2013.

Poliovirus vaccine coverage in Israel is over 90%. The last nine birth cohorts have been vaccinated exclusively with inactivated polio vaccine (IPV). However, between February and July 2013 type 1 wild poliovirus (WPV1) was detected persistently in 10 and intermittently in 8 of 47 environmental surveillance sites in southern and central Israel and in 30 stool samples collected during July from healthy individuals in southern Israel. We report results of sequence and phylogenetic analyses of genes encoding capsid proteins to determine the source and transmission mode of the virus. WPV1 capsid protein 1 nucleotide sequences were most closely related to South Asia (SOAS) cluster R3A polioviruses circulating in Pakistan in 2012 and isolated from Egyptian sewage in December 2012. There was no noticeable geographical clustering within WPV1-positive sites. Uniform codon usage among isolates from Pakistan, Egypt and Israel showed no signs of optimisation or deoptimisation. Bayesian phylogenetic time clock analysis of the entire capsid coding region (2,643 nt) with a 1.1% evolutionary rate indicated that Israeli and Egyptian WPV1-SOAS lineages diverged in September 2012, while Israeli isolates split into two sub-branches after January 2013. This suggests one or more introduction events into Israel with subsequent silent circulation despite high population immunity.

Update on Vaccine-Derived Polioviruses - Worldwide, January 2015-May 2016.

In 1988, the World Health Assembly resolved to eradicate poliomyelitis worldwide (1). One of the main tools used in polio eradication efforts has been the live, attenuated, oral poliovirus vaccine (OPV) (2), an inexpensive vaccine easily administered by trained volunteers. OPV might require several doses to induce immunity, but provides long-term protection against paralytic disease. Through effective use of OPV, the Global Polio Eradication Initiative (GPEI) has brought wild polioviruses to the threshold of eradication (1). However, OPV use, particularly in areas with low routine vaccination coverage, is associated with the emergence of genetically divergent vaccine-derived polioviruses (VDPVs) whose genetic drift from the parental OPV strains indicates prolonged replication or circulation (3). VDPVs can emerge among immunologically normal vaccine recipients and their contacts as well as among persons with primary immunodeficiencies (PIDs). Immunodeficiency-associated VDPVs (iVDPVs) can replicate for years in some persons with PIDs. In addition, circulating vaccine-derived polioviruses (cVDPVs) (3) can emerge in areas with low OPV coverage and can cause outbreaks of paralytic polio. This report updates previous summaries regarding VDPVs (4).

Surveillance Systems to Track Progress Toward Polio Eradication — Worldwide, 2014–2015

Global efforts to eradicate polio began in 1988, and polio-free certification has been achieved in four of the six World Health Organization (WHO) regions. Nigeria was removed from WHOs list of countries with endemic polio in September 2015, achieving an important milestone toward interruption of wild poliovirus (WPV) transmission in the African Region (1). Afghanistan and Pakistan, both in the Eastern Mediterranean Region, were the only countries to report WPV cases in 2015. Previously reported outbreaks caused by WPV importation during 2013-2014 have ended (2,3). The primary means for detecting poliovirus transmission is surveillance for acute flaccid paralysis (AFP) among children aged <15 years (4,5). Stool specimens collected from children with AFP are tested for both WPV and vaccine-derived poliovirus (VDPV) in WHO-accredited laboratories within the Global Polio Laboratory Network (GPLN). In selected locations, AFP surveillance is supplemented with environmental surveillance (testing sewage for poliovirus) (6). Testing of stool and sewage samples includes genomic sequencing to characterize poliovirus isolates; results are used to map poliovirus transmission and identify gaps in AFP surveillance. This report presents poliovirus surveillance data from 2014 and 2015, focusing on the 20 countries in the African Region and six in the Eastern Mediterranean Region that reported a WPV or circulating VDPV (cVDPV) case during 2011-2015, including Guinea, Liberia, and Sierra Leone, which were most affected by the 2014-2015 Ebola virus disease (Ebola) outbreak.

Poliovirus serotype-specific VP1 sequencing primers

The Global Polio Laboratory Network routinely uses poliovirus-specific PCR primers and probes to determine the serotype and genotype of poliovirus isolates obtained as part of global poliovirus surveillance. To provide detailed molecular epidemiologic information, poliovirus isolates are further characterized by sequencing the ~900-nucleotide region encoding the major capsid protein, VP1. It is difficult to obtain quality sequence information when clinical or environmental samples contain poliovirus mixtures. As an alternative to conventional methods for resolving poliovirus mixtures, sets of serotype-specific primers were developed for amplifying and sequencing the VP1 regions of individual components of mixed populations of vaccine-vaccine, vaccine-wild, and wild-wild polioviruses.

Environmental Surveillance for Polioviruses in the Global Polio Eradication Initiative

This article summarizes the status of environmental surveillance (ES) used by the Global Polio Eradication Initiative, provides the rationale for ES, gives examples of ES methods and findings, and summarizes how these data are used to achieve poliovirus eradication. ES complements clinical acute flaccid paralysis (AFP) surveillance for possible polio cases. ES detects poliovirus circulation in environmental sewage and is used to monitor transmission in communities. If detected, the genetic sequences of polioviruses isolated from ES are compared with those of isolates from clinical cases to evaluate the relationships among viruses. To evaluate poliovirus transmission, ES programs must be developed in a manner that is sensitive, with sufficiently frequent sampling, appropriate isolation methods, and specifically targeted sampling sites in locations at highest risk for poliovirus transmission. After poliovirus ceased to be detected in human cases, ES documented the absence of endemic WPV transmission and detected imported WPV. ES provides valuable information, particularly in high-density populations where AFP surveillance is of poor quality, persistent virus circulation is suspected, or frequent virus reintroduction is perceived. Given the benefits of ES, GPEI plans to continue and expand ES as part of its strategic plan and as a supplement to AFP surveillance.