Silvia Peñaranda

An automated genotyping tool for enteroviruses and noroviruses

BACKGROUND Molecular techniques are established as routine in virological laboratories and virus typing through (partial) sequence analysis is increasingly common. Quality assurance for the use of typing data requires harmonization of genotype nomenclature, and agreement on target genes, depending on the level of resolution required, and robustness of methods. OBJECTIVE To develop and validate web-based open-access typing-tools for enteroviruses and noroviruses. STUDY DESIGN An automated web-based typing algorithm was developed, starting with BLAST analysis of the query sequence against a reference set of sequences from viruses in the family Picornaviridae or Caliciviridae. The second step is phylogenetic analysis of the query sequence and a sub-set of the reference sequences, to assign the enterovirus type or norovirus genotype and/or variant, with profile alignment, construction of phylogenetic trees and bootstrap validation. Typing is performed on VP1 sequences of Human enterovirus A to D, and ORF1 and ORF2 sequences of genogroup I and II noroviruses. For validation, we used the tools to automatically type sequences in the RIVM and CDC enterovirus databases and the FBVE norovirus database. RESULTS Using the typing-tools, 785(99%) of 795 Enterovirus VP1 sequences, and 8154(98.5%) of 8342 norovirus sequences were typed in accordance with previously used methods. Subtyping into variants was achieved for 4439(78.4%) of 5838 NoV GII.4 sequences. DISCUSSION AND CONCLUSIONS The online typing-tools reliably assign genotypes for enteroviruses and noroviruses. The use of phylogenetic methods makes these tools robust to ongoing evolution. This should facilitate standardized genotyping and nomenclature in clinical and public health laboratories, thus supporting inter-laboratory comparisons.

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

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.

Characteristics of young infants in whom human parechovirus, enterovirus or neither were detected in cerebrospinal fluid during sepsis evaluations.

Background: Human parechovirus (HPeV) causes central nervous system (CNS) infection in infants. To further understand HPeV CNS infection, we describe its clinical, laboratory and epidemiologic characteristics from a Midwestern US tertiary care center. Because HPeV CNS infections have appeared clinically and seasonally similar to enterovirus (EV) infections, we retrospectively compared characteristics of young infants undergoing sepsis evaluations in whom HPeV, EV or neither were detected in cerebrospinal fluid (CSF). Methods: HPeV real-time reverse-transcription polymerase chain reaction (RT-PCR) assay was performed on frozen nucleic acid extracts of CSF specimens submitted for EV RT-PCR assay from children seen at our hospital in 2009. HPeV genotyping was performed by sequencing of the viral protein 1 region. Clinical data were abstracted from medical records retrospectively for EV-positive, HPeV-positive and age-matched controls in whom neither virus was detected from CSF testing. Results: HPeV was detected in 66 of the 388 (17%) CSF specimens whereas EV was detected in 54 of the 388 (14%) from June through October 2009. Genotyping identified HPeV3 in 51 of the 66 (77%) positive CSF specimens. Males predominated (61%) with the most common presenting symptoms (91%) being fever and irritability. All HPeV-positive patients were <5 months of age. Eight required admission to the pediatric intensive care unit. In multivariate analysis, lower peripheral white blood cell counts with lower absolute lymphocyte count values, higher maximum temperatures, longer fever duration, absence of pleocytosis and longer hospitalization were independently associated with HPeV patients compared with patients with EV or patients negative for both HPeV and EV. Conclusions: Our data indicate that HPeV3, an emerging CNS pathogen of infants in the United States, should be considered in sepsis-like presentation even without CSF pleocytosis. Addition of HPeV RT-PCR to EV RT-PCR assay for CSF specimens of patients <6 months of age could reduce hospital stay and costs while improving clinical management

Increased Activity of Coxsackievirus B1 Strains Associated with Severe Disease among Young Infants in the United States, 2007—2008

BACKGROUND Enterovirus infections are very common and typically cause mild illness, although neonates are at higher risk for severe illness. In 2007, the Centers for Disease Control and Prevention (CDC) received multiple reports of severe neonatal illness and death associated with coxsackievirus B1 (CVB1), a less common enterovirus serotype not previously associated with death in surveillance reports to the CDC. METHODS This report includes clinical, epidemiologic, and virologic data from cases of severe neonatal illness associated with CVB1 reported during the period from 2007 through 2008 to the National Enterovirus Surveillance System (NESS), a voluntary, passive surveillance system. Also included are data on additional cases reported to the CDC outside of the NESS. Virus isolates or original specimens obtained from patients from 25 states were referred to the CDC picornavirus laboratory for molecular typing or characterization. RESULTS During 2007-2008, the NESS received 1079 reports of enterovirus infection. CVB1 accounted for 176 (23%) of 775 reported cases with known serotype, making it the most commonly reported serotype for the first time ever in the NESS. Six neonatal deaths due to CVB1 infection were also reported to the CDC during that time. Phylogenetic analysis of the 2007 and 2008 CVB1 strains indicated that the increase in cases resulted from widespread circulation of a single genetic lineage that had been present in the United States since at least 2001. CONCLUSIONS Healthcare providers and public health departments should be vigilant to the possibility of continuing CVB1-associated neonatal illness, and testing and continued reporting of enterovirus infections should be encouraged.

Detection of human parechovirus (HPeV)-3 in spinal fluid specimens from pediatric patients in the Chicago area

BACKGROUND The human parechoviruses (HPeV) have recently been recognized as important viral pathogens causing various illnesses including sepsis and meningitis in children. However, data from the United States is limited. OBJECTIVES To better understand the epidemiology of HPeV in the United States and its role in pediatric disease through detection and typing of the virus in cerebrospinal fluid specimens. STUDY DESIGN Four hundred and twenty-one spinal fluid samples collected from 373 patients ranging in age from 1 day to 18 years were tested using a real-time reverse transcription-PCR assay. The specimens were originally collected for routine viral and bacterial testing to assist in the diagnosis of meningitis or sepsis. Amplification products of the VP1 region in the virus genome were sequenced to identify the parechovirus type. RESULTS Ten positive specimens were identified from 10 different patients. All ten samples were typed as HPeV3 and were negative for bacteria by culture, and for enterovirus and herpes simplex virus by PCR. All of the HPeV3-infected patients were young infants ranging in age from 6 to 59 days. Infants in whom HPeV3 was detected had significantly decreased peripheral white blood cell counts. Positive specimens were all from the summer and early fall. CONCLUSIONS HPeV3 infection of the central nervous system is found in very young infants in certain years during the summer and early fall, and is associated with leukopenia. Real-time RT-PCR is an effective tool for rapid detection of these infections, and could help prevent unnecessary hospitalization and antibiotic use in HPeV infected infants. More widespread use of this tool in diagnosing HPeV infection would aid in further clarifying the prevalence of this disease in the United States.

Diversity of picornaviruses in rural Bolivia

The family Picornaviridae is a large and diverse group of viruses that infect humans and animals. Picornaviruses are among the most common infections of humans and cause a wide spectrum of acute human disease. This study began as an investigation of acute flaccid paralysis (AFP) in a small area of eastern Bolivia, where surveillance had identified a persistently high AFP rate in children. Stools were collected and diagnostic studies ruled out poliovirus. We tested stool specimens from 51 AFP cases and 34 healthy household or community contacts collected during 2002-2003 using real-time and semi-nested reverse transcription polymerase chain reaction assays for enterovirus, parechovirus, cardiovirus, kobuvirus, salivirus and cosavirus. Anecdotal reports suggested a temporal association with neurological disease in domestic pigs, so six porcine stools were also collected and tested with the same set of assays, with the addition of an assay for porcine teschovirus. A total of 126 picornaviruses were detected in 73 of 85 human individuals, consisting of 53 different picornavirus types encompassing five genera (all except Kobuvirus). All six porcine stools contained porcine and/or human picornaviruses. No single virus, or combination of viruses, specifically correlated with AFP; however, the study revealed a surprising complexity of enteric picornaviruses in a single community.

Acute febrile illness surveillance in a tertiary hospital emergency department: comparison of influenza and dengue virus infections.

In 2009, an increased proportion of suspected dengue cases reported to the surveillance system in Puerto Rico were laboratory negative. As a result, enhanced acute febrile illness (AFI) surveillance was initiated in a tertiary care hospital. Patients with fever of unknown origin for 2-7 days duration were tested for Leptospira, enteroviruses, influenza, and dengue virus. Among the 284 enrolled patients, 31 dengue, 136 influenza, and 3 enterovirus cases were confirmed. Nearly half (48%) of the confirmed dengue cases met clinical criteria for influenza. Dengue patients were more likely than influenza patients to have hemorrhage (81% versus 26%), rash (39% versus 9%), and a positive tourniquet test (52% versus 18%). Mean platelet and white blood cell count were lower among dengue patients. Clinical diagnosis can be particularly difficult when outbreaks of other AFI occur during dengue season. A complete blood count and tourniquet test may be useful to differentiate dengue from other AFIs.

Detection of Echovirus 18 in Human Breast Milk

ABSTRACT We detected enteroviral RNA and cultured infectious virus from a series of banked breast milk samples from the mother of an infant with neonatal sepsis; sequencing of the enterovirus isolate identified it as echovirus type 18. In this case, it is possible that enterovirus transmission occurred through the breast milk.