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ABSTRACT Laboratory diagnosis of orthohantavirus infection is primarily based on serology. However, for a confirmed serological diagnosis, evaluation of a follow-up serum sample is essential, which is time consuming and causes delay. Real-time reverse transcription polymerase chain reaction (RT-PCR) tests, if positive, provide an immediate and definitive diagnosis, and accurately identify the causative agent, where the discriminative nature of serology is suboptimal. We re-evaluated sera from orthohantavirus-suspected clinical cases in the Dutch regions of Twente and Achterhoek from July 2014 to April 2016 for the presence of Puumala orthohantavirus (PUUV), Tula orthohantavirus (TULV), and Seoul orthohantavirus (SEOV) RNA. PUUV RNA was detected in 11% of the total number (n = 85) of sera tested, in 50% of sera positive for anti-PUUV/TULV IgM (n = 16), and in 1.4% of sera negative or indeterminate for anti-PUUV/TULV IgM (n = 69). No evidence was found for the presence of TULV or SEOV viral RNA. Based on these findings, we propose two algorithms to implement real-time RT-PCR testing in routine orthohantavirus diagnostics, which optimally provide clinicians with early confirmed diagnoses and could prevent possible further invasive testing and treatment.IMPORTANCEThe addition of a real-time reverse transcription polymerase chain reaction test to routine orthohantavirus diagnostics may better aid clinical decision making than the use of standard serology tests alone. Awareness by clinicians and clinical microbiologists of this advantage may ultimately lead to a reduction in over-hospitalization and unnecessary invasive diagnostic procedures.

ABSTRACT Since its emergence in the United States in 2014, enterovirus D68 (EV-D68) has been and is associated with severe respiratory diseases and acute flaccid myelitis. Even though EV-D68 has been shown to replicate in different neuronal cells in vitro, it is currently poorly understood which viral factors contribute to the ability to replicate efficiently in cells of the central nervous system and whether this feature is a clade-specific feature. Here, we determined the replication kinetics of clinical EV-D68 isolates from (sub)clades A, B1, B2, B3, and D1 in human neuroblastoma cells (SK-N-SH). Subsequently, we compared sequences to identify viral factors associated with increased viral replication. All clinical isolates replicated in SK-N-SH cells, although there was a large difference in efficiency. Efficient replication of clinical isolates was associated with an amino acid substitution at position 271 of VP1 (E271K), which was acquired during virus propagation in vitro. Recognition of heparan sulfate in addition to sialic acids was associated with increased attachment, infection, and replication. Removal of heparan sulfate resulted in a decrease in attachment, internalization, and replication of viruses with E271K. Taken together, our study suggests that the replication kinetics of EV-D68 isolates in SK-N-SH cells is not a clade-specific feature. However, recognition of heparan sulfate as an additional receptor had a large effect on phenotypic characteristics in vitro. These observations emphasize the need to compare sequences from virus stocks with clinical isolates in order to retrieve phenotypic characteristics from original virus isolates. IMPORTANCE Enterovirus D68 (EV-D68) causes mild to severe respiratory disease and is associated with acute flaccid myelitis since 2014. Currently, the understanding of the ability of EV-D68 to replicate in the central nervous system (CNS), and whether it is associated with a specific clade of EV-D68 viruses or specific viral factors, is lacking. Comparing different EV-D68 clades did not reveal clade-specific phenotypic characteristics. However, we did show that viruses which acquired a cell culture-adapted amino acid substitution in VP1 (E271K) recognized heparan sulfate as an additional receptor. Recognition of heparan sulfate resulted in an increase in attachment, infection, and replication in neuroblastoma cells compared with viruses without this specific amino acid substitution. The ability of EV-D68 viruses to acquire cell culture-adaptive substitutions which have a large effect in experimental settings emphasizes the need to sequence virus stocks.