Chloe McIntyre

Proposals for the classification of human rhinovirus species A, B and C into genotypically assigned types

Human rhinoviruses (HRVs) frequently cause mild upper respiratory tract infections and more severe disease manifestations such as bronchiolitis and asthma exacerbations. HRV is classified into three species within the genus Enterovirus of the family Picornaviridae. HRV species A and B contain 75 and 25 serotypes identified by cross-neutralization assays, although the use of such assays for routine HRV typing is hampered by the large number of serotypes, replacement of virus isolation by molecular methods in HRV diagnosis and the poor or absent replication of HRV species C in cell culture. To address these problems, we propose an alternative, genotypic classification of HRV-based genetic relatedness analogous to that used for enteroviruses. Nucleotide distances between 384 complete VP1 sequences of currently assigned HRV (sero)types identified divergence thresholds of 13, 12 and 13 % for species A, B and C, respectively, that divided inter- and intra-type comparisons. These were paralleled by 10, 9.5 and 10 % thresholds in the larger dataset of >3800 VP4 region sequences. Assignments based on VP1 sequences led to minor revisions of existing type designations (such as the reclassification of serotype pairs, e.g. A8/A95 and A29/A44, as single serotypes) and the designation of new HRV types A101–106, B101–103 and C34–C51. A protocol for assignment and numbering of new HRV types using VP1 sequences and the restriction of VP4 sequence comparisons to type identification and provisional type assignments is proposed. Genotypic assignment and identification of HRV types will be of considerable value in the future investigation of type-associated differences in disease outcomes, transmission and epidemiology.

Proposals for the classification of human rhinovirus species C into genotypically assigned types

Human rhinoviruses (HRVs) are common respiratory pathogens associated with mild upper respiratory tract infections, but also increasingly recognized in the aetiology of severe lower respiratory tract disease. Wider use of molecular diagnostics has led to a recent reappraisal of HRV genetic diversity, including the discovery of HRV species C (HRV-C), which is refractory to traditional virus isolation procedures. Although it is heterogeneous genetically, there has to date been no attempt to classify HRV-C into types analogous to the multiple serotypes identified for HRV-A and -B and among human enteroviruses. Direct investigation of cross-neutralization properties of HRV-C is precluded by the lack of methods for in vitro culture, but sequences from the capsid genes (VP1 and partial VP4/VP2) show evidence for marked phylogenetic clustering, suggesting the possibility of a genetically based system comparable to that used for the assignment of new enterovirus types. We propose a threshold of 13% divergence for VP1 nucleotide sequences for type assignment, a level that classifies the current dataset of 86 HRV-C VP1 sequences into a total of 33 types. We recognize, however, that most HRV-C sequence data have been collected in the VP4/VP2 region (currently 701 sequences between positions 615 and 1043). We propose a subsidiary classification of variants showing > 10% divergence in VP4/VP2, but lacking VP1 sequences, to 28 provisionally assigned types (subject to confirmation once VP1 sequences are determined). These proposals will assist in future epidemiological and clinical studies of HRV-C conducted by different groups worldwide, and provide the foundation for future exploration of type-associated differences in clinical presentations and biological properties.

Comparison of human parechovirus and enterovirus detection frequencies in cerebrospinal fluid samples collected over a 5-year period in edinburgh: HPeV type 3 identified as the most common picornavirus type.

Human enteroviruses (EVs) and more recently parechoviruses (HPeVs) have been identified as the principal viral causes of neonatal sepsis‐like disease and meningitis. The relative frequencies of specific EV and HPeV types were determined over a 5‐year surveillance period using highly sensitive EV and HPeV PCR assays for screening 4,168 cerebrospinal fluid (CSF) specimens collected from hospitalized individuals between 2005 and 2010 in Edinburgh. Positive CSF samples were typed by sequencing of VP1. From the 201 EV and 31 HPeV positive (uncultured) CSF samples on screening, a high proportion of available samples could be directly typed (176/182, 97%). Highest frequencies of EV infections occurred in young adults (n = 43; 8.6%) although a remarkably high proportion of positive samples (n = 98; 46%) were obtained from young infants (<3 months). HPeV infections were seen exclusively in children under the age of 3 months (31/1,105; 2.8%), and confined to spring on even‐numbered years (22% in March 2006, 25% in April 2008, and 22% in March 2010). In contrast, EV infections were distributed widely across the years. Twenty different EV serotypes were detected; E9, E6, and CAV9 being found most frequently, whereas all but one HPeVs were type 3. Over this period, HPeV3 was identified as the most prevalent picornavirus type in CNS‐related infections with similarly high incidences of EV infection frequencies in very young children. The highly sensitive virus typing methods applied in this study will assist further EV and HPeV screening of sepsis and meningitis cases as well as in future molecular epidemiological studies and population surveillance. J. Med. Virol. 83:889–896, 2011.

Analysis of Genetic Diversity and Sites of Recombination in Human Rhinovirus Species C

ABSTRACT Human rhinoviruses (HRVs) are a highly prevalent and diverse group of respiratory viruses. Although HRV-A and HRV-B are traditionally detected by virus isolation, a series of unculturable HRV variants have recently been described and assigned as a new species (HRV-C) within the picornavirus Enterovirus genus. To investigate their genetic diversity and occurrence of recombination, we have performed comprehensive phylogenetic analysis of sequences from the 5′ untranslated region (5′ UTR), VP4/VP2, VP1, and 3Dpol regions amplified from 89 HRV-C-positive respiratory samples and available published sequences. Branching orders of VP4/VP2, VP1, and 3Dpol trees were identical, consistent with the absence of intraspecies recombination in the coding regions. However, numerous tree topology changes were apparent in the 5′ UTR, where >60% of analyzed HRV-C variants showed recombination with species A sequences. Two recombination hot spots in stem-loop 5 and the polypyrimidine tract in the 5′ UTR were mapped using the program GroupingScan. Available HRV-C sequences showed evidence for additional interspecies recombination with HRV-A in the 2A gene, with breakpoints mapping precisely to the boundaries of the C-terminal domain of the encoded proteinase. Pairwise distances between HRV-C variants in VP1 and VP4/VP2 regions fell into two separate distributions, resembling inter- and intraserotype distances of species A and B. These observations suggest that, without serological cross-neutralization data, HRV-C genetic groups may be equivalently classified into types using divergence thresholds derived from distance distributions. The extensive sequence data from multiple genome regions of HRV-C and analyses of recombination in the current study will assist future formulation of consensus criteria for HRV-C type assignment and identification.

Disease burden of the most commonly detected respiratory viruses in hospitalized patients calculated using the disability adjusted life year (DALY) model

Abstract Background The most common acute infections occur in the respiratory tract. Recent discoveries of several novel viruses have markedly increased the repertoire of agents understood to cause presentations of acute respiratory disease. Objectives Further understanding is needed of the relative importance of newly discovered pathogens in the clinical setting to provide clinicians with an indication of appropriate diagnostic and therapeutic targets. To address this, quantification of the disease burden of respiratory viruses in hospitalized patients was undertaken. Study design Disease burden caused by respiratory viruses in hospitalized patients was quantified using the World Health Organization endorsed DALY model. Diagnostic testing results from samples collected over three years for adenovirus (AdV), influenzas A and B, parainfluenza viruses 1, 2 and 3 (PIV-1, -2 and -3), respiratory syncytial virus (HRSV), and previously published retrospective screening for human metapneumovirus, rhinoviruses, and four respiratory coronaviruses were applied to the DALY model. Disability weights were calculated per 1000 hospitalized patients in age banded groups. Results Strikingly different disease burden profiles were observed in children and adults. Adenoviruses were among the leading cause of respiratory presentations in children but not adults. HRSV and influenza A were consistently one of the greatest causes of disease regardless of sampled population. Rhinoviruses and PIV-3 were significant pathogens in all groups except those aged 16–64 years. In immunocompromised patients rhinoviruses were the leading viral cause of disease. Conclusions These analyses provide a framework which can be used to identify where finite resources should be directed in respiratory therapeutics and vaccine development.

High detection frequency and viral loads of human rhinovirus species A to C in fecal samples; diagnostic and clinical implications

Human rhinoviruses (HRVs) can be divided into three species; HRV‐A to HRV‐C. Up to 148 different HRV (sero)types have been identified to date. Because of sequence similarity between 5′‐NCR of HRVs and enteroviruses (EVs), it is problematic to design EV‐specific RT‐PCR assays. The aims of this study were to assess the rate of false‐detection of different rhinoviruses by EV RT‐PCR, and to evaluate the diagnostic and clinical significance of such cross‐reactivity. In vitro RNA transcripts of HRV A‐C created from cDNA templates were quantified spectrophotometrically. Six hundred twenty‐one stool samples screened as part of routine diagnostic for EV, 17 EV‐positive stool samples referred for typing, 288 stool samples submitted for gastroenteritis investigations, and 1,500 CSF samples were included in the study. EV‐specific RT‐PCR detected RNA transcripts of HRV‐A1b, HRV‐B14, and HRV‐Crpat18 but with 10–1,000 reduced sensitivity compared to EV transcripts. Screening fecal samples by EV RT‐PCR identified 13 positive samples identified subsequently as rhinoviruses; a further 26 HRV‐positive samples were identified by nested HRV RT‐PCR. All individuals were hospitalized and presented mostly with diarrhea. A total of 26 HRV types were identified (HRV‐A: 46%; HRV‐B: 13%; HRV‐C: 41%). Results confirm that EV‐specific RT‐PCR can detect HRVs, and at a practical level, identify potential problems of interpretation if fecal samples are used for surrogate screening in cases of suspected viral meningitis. High detection frequencies (10%) and viral loads in stool samples provide evidence for enteric replication of HRV, and its association with enteric disease requires further etiological studies. J. Med. Virol. 84:536–542, 2012.

Development and Assay of RNA Transcripts of Enterovirus Species A to D, Rhinovirus Species A to C, and Human Parechovirus: Assessment of Assay Sensitivity and Specificity of Real-Time Screening and Typing Methods

ABSTRACT Nucleic acid amplification methods such as the PCR have had a major impact on the diagnosis of viral infections, often achieving greater sensitivities and shorter turnaround times than conventional assays and an ability to detect viruses refractory to conventional isolation methods. Their effectiveness is, however, significantly influenced by assay target sequence variability due to natural diversity and rapid sequence changes in viruses that prevent effective binding of primers and probes. This was investigated for a diverse range of enteroviruses (EVs; species A to D), human rhinoviruses (HRVs; species A to C), and human parechovirus (HPeV) in a multicenter assay evaluation using a series of full-length prequantified RNA transcripts. RNA concentrations were quantified by absorption (NanoDrop) and fluorescence methods (RiboGreen) prior to dilution in buffer supplemented with RNase inhibitors and carrier RNA. RNA transcripts were extremely stable, showing minimal degradation after prolonged storage at temperatures between ambient and −20°C and after multiple freeze-thaw cycles. Transcript dilutions distributed to six referral laboratories were screened by real-time reverse transcriptase PCR assays using different primers and probes. All of the laboratories reported high assay sensitivities for EV and HPeV transcripts approaching single copies and similar amplification kinetics for all four EV species. HRV detection sensitivities were more variable, often with substantially impaired detection of HRV species C. This could be accounted for in part by the placement of primers and probes to genetically variable target regions. Transcripts developed in this study provide reagents for the ongoing development of effective diagnostics that accommodate increasing knowledge of genetic heterogeneity of diagnostic targets.

Co-circulation of enteroviruses between apes and humans.

A total of 139 stool samples from wild chimpanzees, gorillas and bonobos in Cameroon and Democratic Republic of Congo (DRC) were screened for enteroviruses (EVs) by reverse transcription PCR. Enterovirus RNA was detected in 10 % of samples, comprising eight from 58 sampled chimpanzees (13.8 %), one from 40 bonobos (2.5 %) and five from 40 gorillas (12.2 %). Three viruses isolated from chimpanzees grouped with human isolate EV-A89 and four (four chimpanzees, one gorilla) represented a newly identified type, EV-A119. These species A virus types overlapped with those circulating in human populations in the same area. The remaining six strains comprised a new species D type, EV-D120, infecting one chimpanzee and four gorillas, and a single EV variant infecting a bonobo that was remarkably divergent from other EVs and potentially constitutes a new enterovirus species. The study demonstrates both the circulation of genetically divergent EV variants in apes and monkeys as well as those shared with local human populations.

Recombination in the evolution of human rhinovirus genomes

Human rhinoviruses (HRV) are highly prevalent human respiratory pathogens that belong to the genus Enterovirus. Although recombination within the coding region is frequent in other picornavirus groups, most evidence of recombination in HRV has been restricted to the 5’ untranslated region. We analysed the occurrence of recombination within published complete genome sequences of members of all three HRV species and additionally compared sequences from HRV strains spanning 14 years. HRV-B and HRV-C showed very little evidence of recombination within the coding region. In contrast, HRV-A sequences appeared to have undergone a large number of recombination events, typically involving whole type groups. This suggests that HRV-A may have been subject to extensive recombination during the period of diversification into types. This study demonstrates the rare and sporadic nature of contemporary recombination of HRV strains and contrasts with evidence of extensive recombination within HRV-A and between members of different species during earlier stages in its evolutionary diversification.

Epidemiology and clinical characteristics of parainfluenza virus 3 outbreak in a Haemato-oncology unit

OBJECTIVES We describe molecular investigations of a large hospital outbreak of parainfluenza virus type 3 (PIV3), in which 32 patients became infected. We outline infection control measures that successfully limited further spread of PIV3 in a Haemato-oncology unit. METHODS Clinical retrospective review of infected haemato-oncology patients was undertaken. PIV3 haemagglutinin sequences from each case (n = 32) and local epidemiologically unlinked controls (n = 53) were compared to identify potential linkage. RESULTS PIV3-infected patients presented with upper (n = 18) and lower (n = 11) respiratory tract infections, 3 showed pyrexia only and one was asymptomatic. All symptomatic patients received antibiotics; bacterial co-infection was confirmed in eleven patients. PIV3 infections were associated with lower mortality than documented previously; three of the PIV3-infected patients died (3/32; 9%). All deaths were associated with relapsed malignancies, and PIV3 was not believed to be the primary cause of death in any of these patients. Sequences from 27 cases clustered closely together, consistent with nosocomial infections from PIV3 circulating within the ward. Factors favouring transmission were high patient turnaround between the day treatment unit and in-patient ward, and limited isolation facilities for immunocompromised and infected patients, especially within the day treatment unit. New infections reduced to baseline levels three days after enhanced infection control interventions were introduced. CONCLUSIONS Molecular epidemiological analysis provided evidence for nosocomial transmission of PIV3 infection that facilitated effective implementation of infection control measures. These were instrumental in restricting further spread of the virus among high-risk patients.