Rapid detection of human parechoviruses in clinical samples by real-time PCR

Benschop, Kimberley; Molenkamp, Richard; Ham, Alwin J. van der; Wolthers, Katja C.; Beld, Marcel

doi:10.1016/j.jcv.2007.10.004

Cited by 100 publications

(41 citation statements)

References 19 publications

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“…The cDNA was subjected to quantitative real-time PCR (Roche) using primers and a probe designed for a 142bp-long conserved 5′ UTR region as described earlier 34 .…”

Section: Methodsmentioning

confidence: 99%

Genomic RNA folding mediates assembly of human parechovirus

et al. 2017

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Assembly of the major viral pathogens of the Picornaviridae family is poorly understood. Human parechovirus 1 is an example of such viruses that contains 60 short regions of ordered RNA density making identical contacts with the protein shell. We show here via a combination of RNA-based systematic evolution of ligands by exponential enrichment, bioinformatics analysis and reverse genetics that these RNA segments are bound to the coat proteins in a sequence-specific manner. Disruption of either the RNA coat protein recognition motif or its contact amino acid residues is deleterious for viral assembly. The data are consistent with RNA packaging signals playing essential roles in virion assembly. Their binding sites on the coat proteins are evolutionarily conserved across the Parechovirus genus, suggesting that they represent potential broad-spectrum anti-viral targets.

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“…The cDNA was subjected to quantitative real-time PCR (Roche) using primers and a probe designed for a 142bp-long conserved 5′ UTR region as described earlier 34 .…”

Section: Methodsmentioning

confidence: 99%

Genomic RNA folding mediates assembly of human parechovirus

et al. 2017

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“…Human enteroviruses and HPeVs are frequent causes of clinical neonatal sepsis syndrome. Due to their replication in the gastrointestinal tract, stool samples are the traditional sources of isolation for these viruses (190)(191)(192)(193). Viral culture of stool specimens, nasal and throat swabs, and cerebrospinal fluid and bronchoalveolar lavage specimens are considered the gold standard for diagnosis, but they typically take several days to weeks to yield positive results and are labor-intensive.…”

Section: Diagnosis Of Sepsis Due To Virusesmentioning

confidence: 99%

“…As a result, investigators have more recently developed real-time PCR-based techniques to identify these viruses in stool, blood, and other body fluids, including cerebrospinal fluid. Different investigators have demonstrated the importance of these viruses in neonatal clinical sepsis and that the yield of the virus was very similar in blood specimens compared to stool specimens in neonates with clinical sepsis (190)(191)(192)(193).…”

Section: Diagnosis Of Sepsis Due To Virusesmentioning

confidence: 99%

Early-Onset Neonatal Sepsis

et al. 2014

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SUMMARY Early-onset sepsis remains a common and serious problem for neonates, especially preterm infants. Group B streptococcus (GBS) is the most common etiologic agent, while Escherichia coli is the most common cause of mortality. Current efforts toward maternal intrapartum antimicrobial prophylaxis have significantly reduced the rates of GBS disease but have been associated with increased rates of Gram-negative infections, especially among very-low-birth-weight infants. The diagnosis of neonatal sepsis is based on a combination of clinical presentation; the use of nonspecific markers, including C-reactive protein and procalcitonin (where available); blood cultures; and the use of molecular methods, including PCR. Cytokines, including interleukin 6 (IL-6), interleukin 8 (IL-8), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α), and cell surface antigens, including soluble intercellular adhesion molecule (sICAM) and CD64, are also being increasingly examined for use as nonspecific screening measures for neonatal sepsis. Viruses, in particular enteroviruses, parechoviruses, and herpes simplex virus (HSV), should be considered in the differential diagnosis. Empirical treatment should be based on local patterns of antimicrobial resistance but typically consists of the use of ampicillin and gentamicin, or ampicillin and cefotaxime if meningitis is suspected, until the etiologic agent has been identified. Current research is focused primarily on development of vaccines against GBS.

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“…Five microliters of cDNA was used to estimate viral copy numbers by using an LC480 real-time PCR machine (Roche Diagnostics). The virus copy numbers per PCR were calculated with a standard curve, as previously described (38). Expression and purification of HPeV capsid proteins.…”

Section: Methodsmentioning

confidence: 99%

Human Memory B Cells Producing Potent Cross-Neutralizing Antibodies against Human Parechovirus: Implications for Prevalence, Treatment, and Diagnosis

Westerhuis

Benschop

Koen

et al. 2015

J Virol

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The family Picornaviridae is a large and diverse group of positive-sense RNA viruses, including human enteroviruses (EVs) and human parechoviruses (HPeVs). The human immune response against EVs and HPeVs is thought to be mainly humoral, and an insufficient neutralizing antibody (Ab) response during infection is a risk factor and can ultimately be life threatening. The accessibility of different antigenic sites and observed cross-reactivity make HPeVs a good target for development of therapeutic human monoclonal antibodies (MAbs). In this study, we generated two different human MAbs specific for HPeV by screening culture supernatants of Ab-producing human B cell cultures for direct neutralization of HPeV1. Both MAbs showed HPeV1-specific neutralization as well as neutralization of HPeV2. One antibody, AM18, cross-neutralized HPeV4, -5, and -6 and coxsackievirus A9 (CV-A9). VP1 capsid protein-specific assays confirmed that AM18 bound VP1 of HPeV1, -2, and -4 with high affinity (11.5 pM). In contrast, the HPeV1-specific MAb AM28, which neutralized HPeV1 even more efficiently than did AM18, showed no cross-reactivity with HPeV3 to -6 or other EVs and did not bind any of the capsid proteins, suggesting that AM28 is specific for a conformation-dependent, nonlinear epitope on the virus. The discovery of MAbs that are cross-reactive between HPeVs may help development of HPeV treatment options with antibodies and vaccine design based on epitopes recognized by these antibodies. IMPORTANCEHPeV infections are widespread among young children and adults, causing a broad range of disease. Infections can be severe and life threatening, while no antiviral treatment is available. Given that the absence of neutralizing Abs is a risk factor for severe disease in infants, treatment of picornavirus infections with MAbs would be a therapeutic option. To study antibody neutralization of HPeV in more detail, we generated two different HPeV1-specific human MAbs. Both MAbs show HPeV1-specific neutralization and cross-neutralized HPeV2. One MAb also cross-neutralized other HPeVs. Surprisingly, this MAb also neutralized CV-A9. These MAbs provide a unique tool for further research and for the diagnosis (antigen detection) and possible treatment of HPeV infections. T he family Picornaviridae is a large and diverse group of positive-sense RNA viruses, which consists of several important human and animal pathogens. This family contains 26 genera, including the Enterovirus and Parechovirus genera. The genus Enterovirus contains Ͼ250 recognized types that can infect humans, including poliovirus (PV), echovirus (E), coxsackie A virus (CV-A), coxsackie B virus (CV-B), and rhinovirus (RV). The genus Parechovirus consists of two species, Ljungan virus and Human parechovirus (HPeV), which at present contains 16 recognized genotypes (1-8). Compared to EV genotypes, which circulate simultaneously, only a few different HPeV genotypes circulate in the human population: HPeV1, -3, and -4 are the most dominant; HPeV5 and -6 are found circulating at ...

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Rapid detection of human parechoviruses in clinical samples by real-time PCR

Cited by 100 publications

References 19 publications

Genomic RNA folding mediates assembly of human parechovirus

Genomic RNA folding mediates assembly of human parechovirus

Early-Onset Neonatal Sepsis

Human Memory B Cells Producing Potent Cross-Neutralizing Antibodies against Human Parechovirus: Implications for Prevalence, Treatment, and Diagnosis

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