Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR.

Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures.

Development of an efficient entire-capsid-coding-region amplification method for direct detection of poliovirus from stool extracts.

Laboratory diagnosis has played a critical role in the Global Polio Eradication Initiative since 1988, by isolating and identifying poliovirus (PV) from stool specimens by using cell culture as a highly sensitive system to detect PV. In the present study, we aimed to develop a molecular method to detect PV directly from stool extracts, with a high efficiency comparable to that of cell culture. We developed a method to efficiently amplify the entire capsid coding region of human enteroviruses (EVs) including PV. cDNAs of the entire capsid coding region (3.9 kb) were obtained from as few as 50 copies of PV genomes. PV was detected from the cDNAs with an improved PV-specific real-time reverse transcription-PCR system and nucleotide sequence analysis of the VP1 coding region. For assay validation, we analyzed 84 stool extracts that were positive for PV in cell culture and detected PV genomes from 100% of the extracts (84/84 samples) with this method in combination with a PV-specific extraction method. PV could be detected in 2/4 stool extract samples that were negative for PV in cell culture. In PV-positive samples, EV species C viruses were also detected with high frequency (27% [23/86 samples]). This method would be useful for direct detection of PV from stool extracts without using cell culture.

Identification of vaccine-derived polioviruses using dual-stage real-time RT-PCR.

Vaccine-derived polioviruses (VDPVs) are associated with polio outbreaks and prolonged infections in individuals with primary immunodeficiencies. VDPV-specific PCR assays for each of the three Sabin oral poliovirus vaccine (OPV) strains were developed, targeting sequences within the VP1 capsid region that are selected for during replication of OPV in the human intestine. Over 2400 Sabin-related isolates and identified 755 VDPVs were screened. Sensitivity of all assays was 100%, while specificity was 100% for serotypes 1 and 3, and 76% for serotype 2. The assays permit rapid, sensitive identification of OPV-related viruses and flag programmatically important isolates for further characterization by genomic sequencing.

Multiple independent emergences of type 2 vaccine-derived polioviruses during a large outbreak in northern Nigeria.

Since 2005, a large poliomyelitis outbreak associated with type 2 circulating vaccine-derived poliovirus (cVDPV2) has occurred in northern Nigeria, where immunization coverage with trivalent oral poliovirus vaccine (tOPV) has been low. Phylogenetic analysis of P1/capsid region sequences of isolates from each of the 403 cases reported in 2005 to 2011 resolved the outbreak into 23 independent type 2 vaccine-derived poliovirus (VDPV2) emergences, at least 7 of which established circulating lineage groups. Virus from one emergence (lineage group 2005-8; 361 isolates) was estimated to have circulated for over 6 years. The population of the major cVDPV2 lineage group expanded rapidly in early 2009, fell sharply after two tOPV rounds in mid-2009, and gradually expanded again through 2011. The two major determinants of attenuation of the Sabin 2 oral poliovirus vaccine strain (A481 in the 5'-untranslated region [5'-UTR] and VP1-Ile143) had been replaced in all VDPV2 isolates; most A481 5'-UTR replacements occurred by recombination with other enteroviruses. cVDPV2 isolates representing different lineage groups had biological properties indistinguishable from those of wild polioviruses, including efficient growth in neuron-derived HEK293 cells, the capacity to cause paralytic disease in both humans and PVR-Tg21 transgenic mice, loss of the temperature-sensitive phenotype, and the capacity for sustained person-to-person transmission. We estimate from the poliomyelitis case count and the paralytic case-to-infection ratio for type 2 wild poliovirus infections that ∼700,000 cVDPV2 infections have occurred during the outbreak. The detection of multiple concurrent cVDPV2 outbreaks in northern Nigeria highlights the risks of cVDPV emergence accompanying tOPV use at low rates of coverage in developing countries.

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.

Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.

We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.

Multiplex PCR method for identifying recombinant vaccine-related polioviruses.

The recent discovery of recombinant circulating vaccine-derived poliovirus (recombinant cVDPV) has highlighted the need for enhanced global poliovirus surveillance to assure timely detection of any future cVDPV outbreaks. Six pairs of Sabin strain-specific recombinant primers were designed to permit rapid screening for VDPV recombinants by PCR.

Emergence of echovirus type 13 as a prominent enterovirus.

In 2001, increased activity of the rarely detected enterovirus echovirus type 13 (E13) was observed in the United States. This article describes the epidemiologic, clinical, and genetic characteristics of E13 activity in the United States in 2001, compared with E13 activity abroad in 2000-2002. In the United States, E13 accounted for 376 (24%) of the 1584 enterovirus isolates reported in 2001 (29% of the reported isolates had a known serotype), compared with 74 isolates reported during 1970-2000. Five states reported aseptic meningitis outbreaks associated with E13, for a total of 521 cases. All characterized E13 isolates from the United States, Europe, Asia, and Oceania recovered in 2000-2002 were at least 95% identical to each other in VP1 capsid gene sequence, but they were genetically distinct from E13 isolates recovered before 2000. Continued surveillance of enteroviruses is important to alert physicians and public health officials to changes in disease trends and to improve efficiencies of clinical intervention.

An echovirus type 33 winter outbreak in New Zealand.

Echovirus type 33 (E33) is a relatively uncommon enterovirus. An E33 outbreak during the winter of 2000 in New Zealand led to 75 virologically-confirmed cases of E33 infection (2.6 cases per 100,000 individuals). Sixty-six (88%) of the 75 patients were aged <30 years, with the highest rates of infection recorded in Maori and Pacific ethnic groups. Overall, 47 (84%) of 56 patients whose cases were analyzed had either aseptic meningitis or encephalitis. Central nervous system involvement was more common after infancy (43 of 45 non-infant patients vs. 4 of 11 infants [relative risk, 2.6; 95% CI, 1.5-4.3]). Two infants died, including a neonate with fulminant hepatitis. Independent of symptom duration, neutrophil-predominant pleocytosis was detected in 17 (41%) of 41 cerebrospinal fluid specimens. Virus isolates could not be definitively typed by antibody neutralization testing but were identified as E33 by partial sequencing of the VP-1 capsid gene. The isolates were closely related to strains from Australia and Oman. Molecular typing, together with a serotype-specific E33 PCR, improved the speed and effectiveness of the outbreak investigation.

Acute flaccid paralysis from echovirus type 33 infection.

During a community echovirus type 33 outbreak, the virus was detected in the feces and cerebrospinal fluid of a 3-year-old boy with right arm weakness that followed a mild nonspecific febrile illness. This is the first time an association between echovirus type 33 infection and acute flaccid paralysis has been reported.

Molecular epidemiology and type-specific detection of echovirus 11 isolates from the Americas, Europe, Africa, Australia, southern Asia and the Middle East.

Echovirus 11 (E11) is among the most commonly isolated human enteroviruses. To examine the range of genetic variation within the E11 serotype, we determined the complete VP1 sequences for 53 geographically dispersed E11 strains isolated in 16 countries from 1953 to 2001. E11 sequences were monophyletic with respect to all other enterovirus serotypes. The sequences clustered into four monophyletic genogroups, A-D; members of each genogroup differed from one another by <20%. Isolates in different genogroups differed from one another by 19-28%. The E11 prototype strain, USA/CA53-Gregory, was the sole member of genogroup B. All recent US isolates were members of one of two discrete lineages within genogroup D. The well-characterized E11 antigenic variant, USA/CA63-Silva, was also a member of genogroup D. Members of genogroups A and C were antigenically similar to USA/CA53-Gregory, as measured by neutralization with anti-Gregory and anti-Silva antisera. Only USA/CA63-Silva was neutralized more efficiently by the anti-Silva antiserum; other genogroup D viruses were Gregory-like or intermediate in their neutralization phenotype. Recent non-US isolates were distributed in genogroups A, C and D. Sequence similarities among genogroup D isolates from North America, Europe, Asia, Australia and North Africa demonstrate that an E11 strain can spread rapidly over a wide geographic area. The aligned sequences were used to develop an E11-specific RT-PCR assay, using degenerate, inosine-containing primers, to amplify all members of all genogroups.