David R. Kilpatrick

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.

ABSTRACT 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.

Serotype-specific identification of enterovirus 71 by PCR☆

BACKGROUND Enterovirus 71 and coxsackievirus A16 are closely related genetically and are causative agents of hand foot and mouth disease. Because enterovirus 71 is more often associated with severe neurological disease, there is a need to rapidly discriminate between enterovirus 71 and coxsackievirus A16 during hand, foot, and mouth disease outbreaks. OBJECTIVES Our goal was to develop and evaluate a serotype-specific reverse transcription-polymerase chain reaction (RT/PCR)-based typing method for enterovirus 71. STUDY DESIGN Two sets of PCR primers were designed to match conserved amino acid intervals of enterovirus 71. One diagnostic primer pair contains deoxyinosine at sites of 4-fold codon degeneracy. A second primer pair was designed for use in sequencing and molecular epidemiology studies. Primer pairs were tested on strains encountered in routine diagnostic samples. RESULTS Using both sets of primers on a panel of 61 prototype enteroviral strains, both primer pairs gave strong positive signals for only enterovirus 71. These primers amplified all enterovirus 71 isolates tested and discriminated between enterovirus 71 and the most closely related enterovirus, coxsackievirus A16. CONCLUSIONS Our RT-PCR assay can be used for specific identification of enterovirus 71 clinical isolates. Furthermore, the 484-bp product of one primer pair has proven useful in sequencing studies to identify distinct genotypes of enterovirus 71.

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.

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.

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.

Multiplex PCR Method for Identifying Recombinant Vaccine-Related Polioviruses

ABSTRACT 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.

Type-Specific Detection of Echovirus 30 Isolates Using Degenerate Reverse Transcriptase PCR Primers

ABSTRACT Following an approach used to specifically identify polioviruses and enterovirus 71, we have developed reverse transcriptase (RT) PCR primers containing mixed-base residues or deoxyinosine at positions of codon degeneracy. These primers permit specific RT-PCR amplification of echovirus 30 (E30) sequences by targeting sites that encode conserved amino acid motifs within the major capsid protein, VP1. All 221 E30 strains tested, isolated in 16 countries over a 44-year period, yielded the predicted 158-bp PCR product. No specific products were obtained by PCR assays containing templates from any of the other 63 EV serotypes. Inosine-containing degenerate primers may be widely applicable to the identification of echovirus serotypes by PCR.

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

ABSTRACT 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.

Some genetic characteristics of sabin-like poliovirus isolated from acute flaccid paralysis cases in Nigeria

A total of 34 sabin strains of the poliovirus isolated from 22 children with 60-day follow-up residual acute flaccid paralysis (AFP) were genetically characterized and screened for any form of recombination. Sequence analysis of the 906-nucleotide capsid showed that all the isolates were similar to their original sabin serotypes, however two of the viruses had drifted in their 3D noncapsid regions toward a sabin-sabin and sabin-nonpolio entero combination. Routine immunization in Nigeria is low and in spite of the increase in the frequency of supplemental immunizations, a lot of children are still inadequately immunized, which may be the reason for our observation in this study. Although we are not dealing with a case of circulating vaccine derived poliovirus (cVDPV) yet, if the above condition persists, the advent of cVDVP may not be too far. There is therefore the need to maintain a high quality mass immunization and sustained routine immunization. Key words : Poliovirus, sequence, crossover, non polio enterovirus, recombination, genome, Sabin-like, vaccine, Nigeria. African Journal of Biotechnology Vol.2(11) 2003: 460-464