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Dive into the research topics where M. Steven Oberste is active.

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Featured researches published by M. Steven Oberste.


Journal of Clinical Microbiology | 2006

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

W. Allan Nix; M. Steven Oberste; Mark A. Pallansch

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.


Journal of Clinical Virology | 2003

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

M. Steven Oberste; William Allan Nix; Kaija Maher; Mark A. Pallansch

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


Journal of Virology | 2003

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

Betty Brown; M. Steven Oberste; Kaija Maher; Mark A. Pallansch

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.


Journal of Virology | 2004

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

M. Steven Oberste; Kaija Maher; Mark A. Pallansch

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.


PLOS Pathogens | 2011

The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling

Amitava Mukherjee; Stefanie A. Morosky; Elizabeth Delorme-Axford; Naomi Dybdahl-Sissoko; M. Steven Oberste; Tianyi Wang; Carolyn B. Coyne

The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR) pathways that converge on the induction of type I interferons (IFNs). Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3Cpro cysteine protease of coxsackievirus B3 (CVB3) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3Cpro, occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3Cpro-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3Cpro cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition.


Clinical Infectious Diseases | 2007

Outbreak of Neurologic Enterovirus Type 71 Disease: A Diagnostic Challenge

Carlos M. Pérez-Vélez; Marsha S. Anderson; Christine C. Robinson; Elizabeth J. McFarland; W. Allan Nix; Mark A. Pallansch; M. Steven Oberste; Mary P. Glodé

BACKGROUND Similar to poliovirus, enterovirus type 71 (EV71) causes severe disease, including aseptic meningitis, encephalitis, acute flaccid paralysis, and acute cardiopulmonary dysfunction. Large epidemics of EV71 infection have been reported worldwide. METHODS After recognition of a cluster of cases of EV71 disease, we reviewed records of patients with EV71 disease who required hospitalization at The Childrens Hospital in Denver, Colorado, from 2003 through 2005. The presence of enterovirus was detected by reverse-transcriptase polymerase chain reaction (PCR) and/or viral culture of specimens from multiple sources, and the virus was typed as EV71 using genetic sequencing. RESULTS Eight cases of EV71 disease were identified in both 2003 and 2005. Fifty-six percent of patients with EV71 disease were < or = 6 months of age (range, 4 weeks to 9 years). All 16 patients had EV71 central nervous system infection. Enterovirus PCR (EV-PCR) of cerebrospinal fluid specimens yielded positive results for only 5 (31.2%) of the 16 patients; all of these patients were < 4 months of age and had less severe disease. However, EV-PCR of upper respiratory tract specimens yielded positive results for 8 (100%) of 8 patients, and EV-PCR of lower gastrointestinal tract specimens yielded positive results for 7 (87.5%) of 8 patients. CONCLUSIONS An outbreak of neurologic EV71 disease occurred in Denver, Colorado, during 2003 and 2005. Likely, EV71 disease remains unrecognized in other parts of the United States, because EV-PCR of cerebrospinal fluid frequently yields negative results. EV-PCR of specimens from the respiratory and gastrointestinal tracts had higher diagnostic yields than did EV-PCR of cerebrospinal fluid. EV71 infection should be considered in young children presenting with aseptic meningitis, encephalitis, acute flaccid paralysis, or acute cardiopulmonary collapse. EV71 infection may be an underrecognized emerging disease in the United States.


Virus Genes | 1992

Conservation of amino-acid sequence motifs in lentivirus Vif proteins

M. Steven Oberste; Matthew A. Gonda

The nonstructural/regulatory genes of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses are believed to play an important role in the replication and pathogenesis of these viruses. In HIV-1 and other lentiviruses, thevif (viral infectivity factor) open reading frame (ORF) (also termedsor orQ in some lentivirus genomes) is located in the central region, overlapping the 3′ end of thepol ORF, but in a different reading frame. Among the lentiviruses, only equine infectious anemia virus lacks avif ORF. The predicted Vif protein sequences from 38 lentiviruses were analyzed for the presence of global and local sequence similarity. The Vif proteins of closely related lentiviruses are highly conserved (HIV-1HXB2:HIV-1mn=91% identity), while those of more distantly related lentirviruses have diverged significantly (HIV-1HXB2: simian immunodeficiency virusmac=30% identity). A search for local sequence similarity revealed that a unifying feature of predicted lentivirus Vif proteins is the presence of at least one of two short, highly conserved sequence motifs, SL(I/V)X4YX9Y and SLQXLA. SLQXLA was present in 34 of 38 lentiviruses examined, while the remaining four lentiviruses had one (three viruses) or two (one virus) substitutions in this motif (of five total substitutions, three were conservative changes). The SL(I/V)X4YX9Y motif was found only in primate lentiviruses and in bovine immunodeficiency-like virus. Based on these findings, we suggest that the locus designationvif be used to denote all lentivirus ORFs previously calledvif, Q, orsor.


Journal of Clinical Microbiology | 2011

Application of TaqMan® Low Density Arrays for Simultaneous Detection of Multiple Respiratory Pathogens

Maja Kodani; Genyan Yang; Laura Conklin; Tatiana Travis; Cynthia G. Whitney; Larry J. Anderson; Stephanie J. Schrag; Thomas H. Taylor; Bernard Beall; Robert F. Breiman; Daniel R. Feikin; M. Kariuki Njenga; Leonard W. Mayer; M. Steven Oberste; Maria Lucia Tondella; Jonas M. Winchell; Stephen Lindstrom; Dean D. Erdman; Barry S. Fields

ABSTRACT The large and growing number of viral and bacterial pathogens responsible for respiratory infections poses a challenge for laboratories seeking to provide rapid and comprehensive pathogen identification. We evaluated a novel application of the TaqMan low-density array (TLDA) cards for real-time PCR detection of 21 respiratory-pathogen targets. The performance of the TLDA was compared to that of individual real-time PCR (IRTP) assays with the same primers and probes using (i) nucleic acids extracted from the 21 pathogen strains and 66 closely related viruses and bacteria and (ii) 292 clinical respiratory specimens. With spiked samples, TLDA cards were about 10-fold less sensitive than IRTP assays. By using 292 clinical specimens to generate 2,238 paired individual assays, the TLDA card exhibited 89% sensitivity (95% confidence interval [CI], 86 to 92%; range per target, 47 to 100%) and 98% specificity (95% CI, 97 to 99%; range per target, 85 to 100%) overall compared to IRTP assays as the gold standard with a threshold cycle (CT ) cutoff of 43. The TLDA card approach offers promise for rapid and simultaneous identification of multiple respiratory pathogens for outbreak investigations and disease surveillance.


Journal of Virology | 2004

RNA Recombination Plays a Major Role in Genomic Change during Circulation of Coxsackie B Viruses

M. Steven Oberste; Silvia Peñaranda; Mark A. Pallansch

ABSTRACT RNA recombination has been shown to occur during circulation of enteroviruses, but most studies have focused on poliovirus. To examine the role of recombination in the evolution of the coxsackie B viruses (CVB), we determined the partial sequences of four genomic intervals for multiple clinical isolates of each of the six CVB serotypes isolated from 1970 to 1996. The regions sequenced were the 5′-nontranslated region (5′-NTR) (350 nucleotides [nt]), capsid (VP4-VP2, 416 nt, and VP1, ∼320 nt), and polymerase (3D, 491 nt). Phylogenetic trees were constructed for each genome region, using the clinical isolate sequences and those of the prototype strains of all 65 enterovirus serotypes. The partial VP1 sequences of each CVB serotype were monophyletic with respect to serotype, as were the VP4-VP2 sequences, in agreement with previously published studies. In some cases, however, incongruent tree topologies suggested that intraserotypic recombination had occurred between the sequenced portions of VP2 and VP1. Outside the capsid region, however, isolates of the same serotype were not monophyletic, indicating that recombination had occurred between the 5′-NTR and capsid, the capsid and 3D, or both. Almost all clinical isolates were recombinant relative to the prototype strain of the same serotype. All of the recombination partners appear to be members of human enterovirus species B. These results suggest that recombination is a frequent event during enterovirus evolution but that there are genetic restrictions that may influence recombinational compatibility.


Pediatric Infectious Disease Journal | 2006

Neonatal enterovirus infections reported to the national enterovirus surveillance system in the United States, 1983-2003.

Nino Khetsuriani; Ashley C. LaMonte; M. Steven Oberste; Mark A. Pallansch

Background: Neonatal enterovirus (EV) infections lead to a wide range of clinical manifestations, from mild febrile illness to severe, sometimes fatal, sepsislike disease. Methods: To determine the relationship of EV serotypes with the risk of neonatal infection and its fatal outcome, we analyzed data reported to the National Enterovirus Surveillance System (NESS) during 1983–2003. Results: Of the 26,737 EV detections reported during this period, neonates accounted for 2544 (11.4% of those with known age). Serotypes most commonly isolated from neonates included echovirus (E) 11 (14.0% of EV with known serotype), coxsackievirus (CV) B2 (8.9%), CVB5 (7.5%), E6, E9 and CVB4 (6.8% each). CVB1-4, E11, and E25 were significantly more common, whereas CVA16, E4, E9, E21, E30, and human parechovirus 1 (formerly E22) were less common among neonates than among persons aged ≥1 month. Fatal outcome was noted for 3.3% of reports, with neonates at a higher risk of death than persons aged ≥1 month (11.5% versus 2.5%; odds ratio [OR] 5.1; 95% confidence interval [CI] = 3.3–7.8). Neonates infected with CVB4 were at a higher risk of death (OR 6.5; 95% CI = 2.4–17.7) than those infected with other EV. Conclusion: EV are important neonatal pathogens associated with high risk of infection and death. Because of the limitations of the NESS (incomplete reporting, limited clinical data, bias towards more severe and younger cases), additional studies are needed to better evaluate the role of different EV in neonatal infections.

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Mark A. Pallansch

Centers for Disease Control and Prevention

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W. Allan Nix

Centers for Disease Control and Prevention

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William C. Weldon

Centers for Disease Control and Prevention

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Kaija Maher

Centers for Disease Control and Prevention

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Cara C. Burns

Centers for Disease Control and Prevention

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David R. Kilpatrick

Centers for Disease Control and Prevention

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Silvia Peñaranda

Centers for Disease Control and Prevention

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Ondrej Mach

World Health Organization

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Dean D. Erdman

Centers for Disease Control and Prevention

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