Detection, Identification and Molecular Variation of Human Enteroviruses

Picornaviruses are the most common human viruses and the identification of the picornaviruses is nowadays based on molecular techniques, for example, reverse transcriptase polymerase chain reaction (RT-PCR). One aim of this thesis was to improve the identification of picornaviruses, especially rhino- and enteroviruses, with a real-time assay format and, also, to improve the differentiation of the viruses with genus-specific locked nucleic acid (LNA) probes. Another aim was to identify and study the causative agent of the enterovirus epidemics that appeared in Finland during seasons 2008-2010. In this thesis, the first version of picornavirus qRT-PCR with a melting curve analysis was used in a study of rhinovirus transmission within families with a rhinovirus positive index child where rhinovirus infection was monitored in all family members. In conclusion, rhinoviruses spread effectively within families causing mostly symptomatic infections in children and asymptomatic infections in adults. To improve the differentiation between rhino- and enterovirus the picornavirus qRT-PCR was modified with LNA-incorporated probes. The LNA probes were validated with picornavirus prototypes and different clinical specimen types. The LNA probe-based picornavirus qRT-PCR was able to differentiate all rhino- and enteroviruses correctly, which makes it suitable for diagnostic use. Moreover, in this thesis enterovirus outbreaks were studied with a well-observed method to create a strain-specific qRT-PCR from the typing region VP1 protein. In a hand-foot-and-mouth-disease (HFMD) outbreak in 2008, the causative agent was identified as CV-A6 and when the molecular evolution of the new HFMD CV-A6 strain was studied it was found that CV-A6 was the emerging agent for HFMD and onychomadesis. Furthermore, unusual E-30 meningitis epidemics that apeared during seasons 2009 and 2010 were studied with strain-specific qRT-PCR. The E-30 affected mostly adolescents and was probably spread in sports teams.Siirretty Doriast

Genetic characterization of human coxsackievirus A6 variants associated with atypical hand, foot and mouth disease: a potential role of recombination in emergence and pathogenicity

Human coxsackievirus A6 (CVA6) is an enterically transmitted enterovirus. Until recently, CVA6 infections were considered as being of minor clinical significance, and only rarely aetiologically linked with hand, foot and mouth disease (HFMD) associated with other species A enteroviruses (particularly EV71 and CVA16). From 2008 onwards, however, CVA6 infections have been associated with several outbreaks worldwide of atypical HFMD (aHFMD) accompanied by a varicelliform rash. We recently reported CVA6-associated eczema herpeticum occurring predominantly in children and young adults in Edinburgh in January and February 2014. To investigate genetic determinants of novel clinical phenotypes of CVA6, we genetically characterized and analysed CVA6 variants associated with eczema herpeticum in Edinburgh in 2014 and those with aHFMD in CAV isolates collected from 2008. A total of eight recombinant forms (RFs) have circulated worldwide over the past 10 years, with the particularly recent appearance of RF-H associated with eczema herpeticum cases in Edinburgh in 2014. Comparison of phylogenies and divergence of complete genome sequences of CVA6 identified recombination breakpoints in 2A-2C, within VP3, and between 5' untranslated region and VP1. A Bayesian temporal reconstruction of CVA6 evolution since 2004 provided estimates of dates and the actual recombination events that generated more recently appearing recombination groups (RF-E, -F, -G and -H). Associations were observed between recombination groups and clinical presentations of herpangina, aHFMD and eczema herpeticum, but not with VP1 or other structural genes. These observations provided evidence that NS gene regions may potentially contribute to clinical phenotypes and outcomes of CVA6 infection

Simultaneous Detection and Differentiation of Human Rhino- and Enteroviruses in Clinical Specimens by Real-Time PCR with Locked Nucleic Acid Probes

Abstract Human rhinoviruses (HRVs) and human enteroviruses (HEVs) are significant respiratory pathogens. While HRV infections are restricted to the respiratory tract, HEV infections may spread to secondary target organs. The method of choice for sensitive specific detection of these viruses is reverse transcription (RT)-PCR with primers targeting the conserved 5' noncoding region of the viral RNA. On the other hand, sequence similarities between HRVs and HEVs complicate their differential detection. In this study, we describe the use of locked nucleic acid (LNA) analogues in short double-dye probes which contained only two selectively HRV- or HEV-specific bases. The double-stranded DNA dye BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinolinium chloride) was used with the LNA probes in a tricolor real-time PCR assay to allow specific detection of HRVs (probes labeled with 6-carboxyfluorescein [FAM] [green]) and HEVs (Cy5 [red]) with additional melting curve analysis (BOXTO [yellow]). The functionality of the probes was validated in PCR and RT-PCR assays using plasmids containing viral cDNA, quantified viral RNA transcripts, cultivated rhino- and enterovirus prototypes, and clinical specimens. Of 100 HRV and 63 HEV prototypes, the probes correctly identified all HEVs except one that produced only a BOXTO signal. Among 118 clinical specimens with sequencing results, concordant results were obtained for 116 specimens. Two specimens were reactive with both probes, but sequencing yielded only a single virus. Real-time PCR with LNA probes allowed sensitive group-specific identification of HRVs and HEVs and would enable relative copy number determination. The assay is suitable for rapid and accurate differential detection of HRVs and HEVs in a diagnostic laboratory setting. </div

Genetic characterisation of human Coxsackievirus A6 variants associated with atypical hand, foot and mouth disease; a potential role of recombination in emergence and pathogenicity

Human coxsackievirus A6 (CVA6) is an enterically transmitted enterovirus. Until recently, CVA6 infections were considered as being of minor clinical significance, and only rarely aetiologically linked with hand, foot and mouth disease (HFMD) associated with other species A enteroviruses (particularly EV71 and CVA16). From 2008 onwards, however, CVA6 infections have been associated with several outbreaks worldwide of atypical HFMD (aHFMD) accompanied by a varicelliform rash. We recently reported CVA6-associated eczema herpeticum occurring predominantly in children and young adults in Edinburgh in January and February 2014. To investigate genetic determinants of novel clinical phenotypes of CVA6, we genetically characterized and analysed CVA6 variants associated with eczema herpeticum in Edinburgh in 2014 and those with aHFMD in CAV isolates collected from 2008. A total of eight recombinant forms (RFs) have circulated worldwide over the past 10 years, with the particularly recent appearance of RF-H associated with eczema herpeticum cases in Edinburgh in 2014. Comparison of phylogenies and divergence of complete genome sequences of CVA6 identified recombination breakpoints in 2A–2C, within VP3, and between 5′ untranslated region and VP1. A Bayesian temporal reconstruction of CVA6 evolution since 2004 provided estimates of dates and the actual recombination events that generated more recently appearing recombination groups (RF-E, -F, -G and -H). Associations were observed between recombination groups and clinical presentations of herpangina, aHFMD and eczema herpeticum, but not with VP1 or other structural genes. These observations provided evidence that NS gene regions may potentially contribute to clinical phenotypes and outcomes of CVA6 infection

Human Metapneumovirus Infections in Children

Age-related incidence and effects of these infections are highest among children <2 years of age

Human Metapneumovirus Infections in Children

Age-related incidence and effects of these infections are highest among children <2 years of age