Mark A. Pallansch

Sensitive, Seminested PCR Amplification of VP1 Sequences for Direct Identification of All Enterovirus Serotypes from Original Clinical Specimens

ABSTRACT A reverse transcription-seminested PCR (RT-snPCR) assay was developed for the detection and identification of enterovirus (EV) RNA in clinical specimens. Three conserved protein motifs were identified by aligning the VP3 and VP1 sequences of prototype EV strains. Consensus degenerate primers were designed from a conserved VP3 motif and a distal VP1 motif for the first PCR. Consensus-degenerate hybrid oligonucleotide primers were designed from an internal VP1 motif and used with the same distal VP1 motif for the second, seminested PCR step. The primers were designed for broad target specificity and amplified all recognized and proposed EV serotypes and other antigenic variant strains tested. The VP1 RT-snPCR assay was slightly more sensitive than our in-house EV 5′ nontranslated region RT-snPCR assay, detecting as few as 10 RNA copies per reaction. As an example application, the VP1 RT-snPCR assay was used to identify EVs in clinical specimens. A product of the expected size was successfully amplified and sequenced from cerebrospinal fluid; serum; stool suspensions; and nasopharyngeal, eye, and rectal swab specimens, allowing unambiguous identification of the infecting virus in all cases. The VP1 sequences derived from the RT-snPCR products allow rapid phylogenetic and molecular epidemiologic analysis of strains circulating during the EV season and comparison with EV sequences from past seasons or from different locations around the world.

Deaths of Children during an Outbreak of Hand, Foot, and Mouth Disease in Sarawak, Malaysia: Clinical and Pathological Characteristics of the Disease

From April through June 1997, 29 previously healthy children aged <6 years (median, 1.5 years) in Sarawak, Malaysia, died of rapidly progressive cardiorespiratory failure during an outbreak of hand, foot, and mouth disease caused primarily by enterovirus 71 (EV71). The case children were hospitalized after a short illness (median duration, 2 days) that usually included fever (in 100% of case children), oral ulcers (66%), and extremity rashes (62%). The illness rapidly progressed to include seizures (28%), flaccid limb weakness (17%), or cardiopulmonary symptoms (of 24 children, 17 had chest radiographs showing pulmonary edema, and 24 had echocardiograms showing left ventricular dysfunction), resulting in cardiopulmonary arrest soon after hospitalization (median time, 9 h). Cardiac tissue from 10 patients showed normal myocardium, but central nervous system tissue from 5 patients showed inflammatory changes. Brain-stem specimens from 2 patients were available, and both specimens showed extensive neuronal degeneration, inflammation, and necrosis, suggesting that a central nervous system infection was responsible for the disease, with the cardiopulmonary dysfunction being neurogenic in origin. EV71 and possibly an adenovirus, other enteroviruses, or unknown cofactors are likely responsible for this rapidly fatal disease.

Geographic distribution of wild poliovirus type 1 genotypes

Determination of the patterns of genomic variation among RNA virus isolates is a powerful approach for establishing their epidemiologic interrelationships. The standard technique for such studies, ribonuclease T1 oligonucleotide fingerprinting, can detect similarities only among very closely related isolates. The rapid evolution of the poliovirus genome during transmission in humans requires the application of alternate methods to identify more distant relationships. To obtain a substantially broader view of the distribution of wild poliovirus type 1 genotypes in nature, we compared 150 bases of genomic sequence information (encoding parts of the capsid protein VP1 and the noncapsid protein 2A) from 62 isolates obtained from poliomyelitis patients in five continents. The partial sequence information allowed us to (1) identify numerous geographic foci of endemic circulation of wild type 1 polioviruses, (2) reveal previously unsuspected links between cases in distant communities, (3) monitor the displacement from the environment of preexisting polioviruses by viruses from other regions, and (4) recognize the recombinant (vaccine-wild; wild-wild) origins of some epidemic polioviruses.

Improved molecular identification of enteroviruses by RT-PCR and amplicon sequencing.

The use of diagnostic RT-PCR has improved the ability to rapidly detect enteroviruses (EV) in some clinical specimens; however, these methods are not able to identify which enterovirus has been detected. Although the identification of EV serotype rarely impacts the clinical management of the infected patient, typing can provide valuable epidemiologic information, especially in outbreak investigations and in disease association studies. While the gold standard for EV identification is the neutralization of isolates in cell culture using standardized antiserum pools, this method has several disadvantages: (1) it is time-consuming and labor-intensive; (2) only 40 of 64 serotypes are represented in the pools; (3) the world supply of antisera (made more than 30 years ago) is limited; and (4) the technique is confounded by antigenic variants and strains that tend to aggregate (Oberste et al., 2000, 1999a). 2. Primer selection

Complete Genomic Sequencing Shows that Polioviruses and Members of Human Enterovirus Species C Are Closely Related in the Noncapsid Coding Region

ABSTRACT The 65 human enterovirus serotypes are currently classified into five species: Poliovirus (3 serotypes), Human enterovirus A (HEV-A) (12 serotypes), HEV-B (37 serotypes), HEV-C (11 serotypes), and HEV-D (2 serotypes). Coxsackie A virus (CAV) serotypes 1, 11, 13, 15, 17, 18, 19, 20, 21, 22, and 24 constitute HEV-C. We have determined the complete genome sequences for the remaining nine HEV-C serotypes and compared them with the complete sequences of CAV21, CAV24, and the polioviruses. The viruses were most diverse in the capsid region (4 to 36% amino acid difference). A high degree of capsid sequence conservation (96% amino acid identity) suggests that CAV15 and CAV18 should be classified as strains of CAV11 and CAV13, respectively. In the 3CD region, CAV1, CAV19, and CAV22 differed from one another by only 1.2 to 1.4% and CAV11, CAV13, CAV17, CAV20, CAV21, CAV24, and the polioviruses differed from one another by only 1.2 to 3.6%. The two groups, however, differed from one another by 14.6 to 16.2%. The polioviruses as a group were monophyletic only in the capsid region. Only one group of serotypes (CAV1, CAV19, and CAV22) was consistently monophyletic in multiple genome regions. Incongruities among phylogenetic trees based on different genome regions strongly suggest that recombination has occurred between the polioviruses, CAV11, CAV13, CAV17, and CAV20. The close relationship among the polioviruses and CAV11, CAV13, CAV17, CAV20, CAV21, and CAV24 and the uniqueness of CAV1, CAV19, and CAV22 suggest that revisions should be made to the classification of these viruses.

Evidence for Frequent Recombination within Species Human Enterovirus B Based on Complete Genomic Sequences of All Thirty-Seven Serotypes

ABSTRACT The species Human enterovirus B (HEV-B) in the family Picornaviridae consists of coxsackievirus A9; coxsackieviruses B1 to B6; echoviruses 1 to 7, 9, 11 to 21, 24 to 27, and 29 to 33; and enteroviruses 69 and 73. We have determined complete genome sequences for the remaining 22 HEV-B serotypes whose sequences were not represented in public databases and analyzed these in conjunction with previously available complete sequences in GenBank. Members of HEV-B were monophyletic relative to all other human enterovirus species in all regions of the genome except in the 5′-nontranslated region (NTR), where they are known to cluster with members of HEV-A. Within HEV-B, phylogenies constructed from the structural (P1) and nonstructural regions of the genome (P2 and P3) are incongruent, suggesting that recombination had occurred. Similarity plots and bootscanning analysis across the complete genome identified multiple sites at which the phylogeny of a given strains sequence shifted, indicating potential recombination points. These points are distributed in the 5′-NTR and throughout P2 and P3, but no sites with >80% bootstrap support were identified within the capsid. Individual sequence comparisons and phylogenetic analyses suggest that members of HEV-B have recombined with one another on multiple occasions, resulting in a complex mosaic of sequences derived from multiple parental viruses in the nonstructural regions of the genome. We conclude that RNA recombination is a common mechanism for enterovirus evolution and that recombination within the nonstructural regions of the genome (P2 and P3) has been observed only among members of the same species.

Circulating vaccine-derived polioviruses: current state of knowledge

Within the past 4 years, poliomyelitis outbreaks associated with circulating vaccine-derived polioviruses (cVDPVs) have occurred in Hispaniola (2000-01), the Philippines (2001), and Madagascar (2001-02). Retrospective studies have also detected the circulation of endemic cVDPV in Egypt (1988-93) and the likely localized spread of oral poliovirus vaccine (OPV)-derived virus in Belarus (1965-66). Gaps in OPV coverage and the previous eradication of the corresponding serotype of indigenous wild poliovirus were the critical risk factors for all cVDPV outbreaks. The cVDPV outbreaks were stopped by mass immunization campaigns using OPV. To increase sensitivity for detecting vaccine-derived polioviruses (VDPVs), in 2001 the Global Polio Laboratory Network implemented additional testing requirements for all poliovirus isolates under investigation. This approach quickly led to the recognition of the Philippines and Madagascar cVDPV outbreaks, but of no other current outbreaks. The potential risk of cVDPV emergence has increased dramatically in recent years as wild poliovirus circulation has ceased in most of the world. The risk appears highest for the type 2 OPV strain because of its greater tendency to spread to contacts. The emergence of cVDPVs underscores the critical importance of eliminating the last pockets of wild poliovirus circulation, maintaining universally high levels of polio vaccine coverage, stopping OPV use as soon as it is safely possible to do so, and continuing sensitive poliovirus surveillance into the foreseeable future. Particular attention must be given to areas where the risks for wild poliovirus circulation have been highest, and where the highest rates of polio vaccine coverage must be maintained to suppress cVDPV emergence.

Circulation of Endemic Type 2 Vaccine-Derived Poliovirus in Egypt from 1983 to 1993

ABSTRACT From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5′ untranslated region (5′ UTR) and noncapsid- 3′ UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide.

COMPLETE NUCLEOTIDE SEQUENCE OF ENTEROVIRUS 71 IS DISTINCT FROM POLIOVIRUS

Enterovirus 71 (EV71) is capable of causing paralytic disease indistinguishable from poliomyelitis due to poliovirus. To determine the relationship of EV71 to poliovirus and other enteroviruses, two strains of EV71 have been cloned and sequenced. The EV71 strains had only 46% amino acid identity with the polioviral P1 capsid region and 55% with the entire polyprotein. There were no regions of high similarity that might account for their respective ability to cause paralytic disease. The two strains, a neurovirulent isolate (EV71/7423/MS/87) and the prototype strain (EV71/BrCr), share 81% nucleotide identity and 95% amino acid identity. Sequence comparisons in the coding region between the two EV71 strains and other picornaviruses indicate that EV71, coxsackievirus A16 and coxsackievirus A2 comprise a distinct genetic group within the enteroviruses.

Hospital-associated outbreak of Middle East Respiratory Syndrome Coronavirus: A serologic, epidemiologic, and clinical description

Novel serological tests allowed for the detection of otherwise unrecognized cases of Middle East respiratory syndrome coronavirus infection among contacts in a hospital-associated respiratory illness outbreak in Jordan in April 2012, resulting in a total of 9 test-positive cases.