Cassandra E. Faux

Detection of novel influenza A(H1N1) virus by real-time RT-PCR

Accurate and rapid diagnosis of novel influenza A(H1N1) infection is critical for minimising further spread through timely implementation of antiviral treatment and other public health based measures. In this study we developed two TaqMan-based reverse transcription PCR (RT-PCR) methods for the detection of novel influenza A(H1N1) virus targeting the haemagglutinin and neuraminidase genes. The assays were validated using 152 clinical respiratory samples, including 61 Influenza A positive samples, collected in Queenland, Australia during the years 2008 to 2009 and a further 12 seasonal H1N1 and H3N2 influenza A isolates collected from years 2000 to 2002. A wildtype swine H1N1 isolate was also tested. RNA from an influenza A(H1N1) virus isolate (Auckland, 2009) was used as a positive control. Overall, the results showed that the RT-PCR methods were suitable for sensitive and specific detection of novel influenza A(H1N1) RNA in human samples.

Do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections?

Abstract Background Human rhinoviruses (HRVs) are often concurrently detected with other viruses found in the respiratory tract because of the high total number of HRV infections occurring throughout the year. This feature has previously relegated HRVs to being considered passengers in acute respiratory infections. HRVs remain poorly characterized and are seldom included as a target in diagnostic panels despite their pathogenic potential, infection-associated healthcare expenditure and relatively unmoderated elicitation of an antiviral state. Objectives To test the hypothesis that respiratory viruses are proportionately more or less likely to co-occur, particularly the HRVs. Study design Retrospective PCR-based analyses of 1247 specimens for 17 viruses, including HRV strains, identified 131 specimens containing two or more targets. We investigated the proportions of co-detections and compared the proportion of upper vs. lower respiratory tract presentations in the HRV positive group. Both univariate contingency table and multivariate logistic regression analyses were conducted to identify trends of association among the viruses present in co-detections. Results Many of the co-detections occurred in patterns. In particular, HRV detection was associated with a reduced probability of detecting human adenoviruses, coronaviruses, bocavirus, metapneumovirus, respiratory syncytial virus, parainfluenza virus, influenza A virus, and the polyomaviruses KIPyV and WUPyV (p ≤0.05). No single HRV species nor cluster of particular strains predominated. Conclusions HRVs were proportionately under-represented among viral co-detections. For some period, HRVs may render the host less likely to be infected by other viruses.

Molecular characterization and distinguishing features of a novel human rhinovirus (HRV) C, HRVC-QCE, detected in children with fever, cough and wheeze during 2003

Abstract Background Human rhinoviruses (HRVs) are associated with more acute respiratory tract infections than any other viral group yet we know little about viral diversity, epidemiology or clinical outcome resulting from infection by strains, in particular the recently identified HRVs. Objectives To determine whether HRVC-QCE was a distinct HRV-C strain, by determining its genome and prevalence, by cataloguing genomic features for strain discrimination and by observing clinical features in positive patients. Study design Novel real-time RT-PCRs and retrospective chart reviews were used to investigate a well-defined population of 1247 specimen extracts to observe the prevalence and the clinical features of each HRV-QCE positive case from an in- and out-patient pediatric, hospital-based population during 2003. An objective illness severity score was determined for each HRVC-QCE positive patient. Results Differences in overall polyprotein and VP1 binding pocket residues and the predicted presence of a cis-acting replication element in 1B defined HRVC-QCE as a novel HRV-C strain. Twelve additional HRVC-QCE detections (1.0% prevalence) occurred among infants and toddlers (1–24 months) suffering mild to moderate illness, including fever and cough, who were often hospitalized. HRVC-QCE was frequently detected in the absence of another virus and was the only virus detected in three (23% of HRVC-QCE positives) children with asthma exacerbation and in two (15%) toddlers with febrile convulsion. Conclusions HRVC-QCE is a newly identified, genetically distinct HRV strain detected in hospitalized children with a range of clinical features. HRV strains should be independently considered to ensure we do not overestimate the HRVs in asymptomatic illness.

Community-Wide, Contemporaneous Circulation of a Broad Spectrum of Human Rhinoviruses in Healthy Australian Preschool-Aged Children During a 12-Month Period

Abstract Human rhinovirus (HRV) replication triggers exacerbation of asthma and causes most acute respiratory illnesses (ARIs), which may manifest as influenza-like illness. The recent assignment of 60 previously unknown HRV types to a third HRV species, Human rhinovirus C, raised questions about the prevalence of these picornavirus types in the community, the extent of HRV diversity at a single site, and whether the HRVs have an equally diverse clinical impact on their hosts. We quantified HRV diversity, and there was no clinical impact attributable to HRV species and genotypes among a community population of preschool-aged children with ARI who provided respiratory samples during 2003. All HRV species were represented among 138 children with ARI, and 74 distinct HRV types were cocirculating. Fever accompanied 32.8% of HRV-positive ARI cases. HRVs were less likely than DNA viruses to be codetected with another virus, suggesting virus interference at the community level, demonstrated by the inverse correlation between influenza virus detection and HRV detection.

Usefulness of published PCR primers in detecting human rhinovirus infection

We conducted a preliminary comparison of the relative sensitivity of a cross-section of published human rhinovirus (HRV)–specific PCR primer pairs, varying the oligonucleotides and annealing temperature. None of the pairs could detect all HRVs in 2 panels of genotyped clinical specimens; >1 PCR is required for accurate description of HRV epidemiology.

Prior evidence of putative novel rhinovirus species, Australia.

We appreciate the enthusiasm for our recent publication highlighting the global distribution of a long-unrecognized third clade of rhinoviruses. Robust, sequence-based clock estimates with associated confidence limits indicate that these viruses have been circulating for hundreds of years (1), consistent with the presence of such viruses in historic samples. As isolates from various collections are analyzed in informative regions (e.g., virus protein [VP] 4/2 or VP1), we will undoubtedly find examples in which human rhinoviruses (HRVs) could have been classified as members of the new species HRV-C but were not because the characteristics that define HRV-C were not yet appreciated or because only noncoding sequences had been analyzed. Indeed, we anticipate that waxing interest in HRVs may well lead to the discovery of additional clades. There has been discussion in the field as to whether the novel sequences represent a sublineage HRV-A2 of the classified species HRV-A (2,3), as Mackay et al. had proposed, or whether they should be considered as representatives of a third species of HRV (4,5). The International Committee on Taxonomy of Viruses (ICTV) is charged with the recognition and naming of taxonomic entities. Thus, we provisionally designated our sequences as a novel clade distinct from HRV-A and HRV-B (4) and submitted a proposal to ICTV with data supporting the recognition of HRV-C as a third species of rhinovirus. The proposal was recently approved by the ICTV Study Group on Picornaviruses (Europic May 2008 meeting in Sitges, Spain). Irrespective of taxonomic discourse, we agree with Mackay and colleagues that molecular analyses of as-yet-uncultured HRVs are fascinating and have potential to reveal unexpected insights into the role of HRVs in disease.

Human rhinovirus C in adult haematopoietic stem cell transplant recipients with respiratory illness

Abstract Background A previously unidentified species of human rhinovirus, HRV-C, was described in 2006 in association with lower respiratory tract infection (LRTI). Features of infection in immunosuppressed adults are poorly characterised. Objectives This study aims to determine the epidemiology of HRV-C in haematopoietic stem cell transplant (HSCT) recipients in a single centre. Study design A prospective cohort study of all HSCT recipients admitted to Westmead Hospital, Westmead, Australia from 1 July 2005 to 30 September 2007 was undertaken. Nose/throat samples were collected from all patients at the time of admission and patients developing pre-defined symptoms and/or signs of respiratory infection during the admission. Samples were processed and tested for rhinoviruses and 14 other respiratory viruses using nucleic acid-based methods, immunofluorescence and culture. HRV genotyping was performed by sequencing a region of the rhinovirus 5′ untranslated region (UTR). Clinical data on each episode were collected prospectively. Results HRVs were identified in 24 episodes: 8% of 299 episodes of clinically- defined respiratory infections and 39% of 61 episodes in which respiratory viruses were detected. HRV-C was most frequent (HRV-C: nine, HRV-A: eight and HRV-B: two). Seven episodes of HRV-C, five with pneumonia, occurred within 100 days of HSCT. Co-pathogens were frequent. Conclusions The newly described HRV-C was the most common rhinovirus group detected in HSCT recipients with respiratory infection, with co-pathogens being frequent. Further research is required to understand the activity and pathogenicity of this virus in HSCT recipients.

A simple approach for preparing real-time PCR positive reaction controls for rare or emerging viruses.

BACKGROUND Laboratories often have difficulties obtaining positive control material for polymerase chain reaction (PCR) diagnosis of rare or emerging viruses. This is particularly problematic during outbreaks caused by emerging infectious diseases, when delays can impede the public health response. OBJECTIVES The aim of this study was to develop a simple approach for preparing real-time PCR positive reaction controls for rare or emerging viruses. STUDY DESIGN We describe a universal method for preparing PCR positive reaction controls (Uni-Control), which uses two synthetic control oligonucleotides and irrelevant viral nucleic acid as an initiator template. In this study, we prepared Uni-Controls for novel influenza A(H1N1) and human metapneumovirus (HMPV) RT-PCR assays. Parainfluenza type 2 virus RNA and equine herpes virus DNA were used as initiator templates. RESULTS Using the Uni-Controls, characteristic sigmoidal real-time PCR amplification curves were observed in the influenza A(H1N1) and HMPV RT-PCR assays. Comparable cycle threshold values were observed in both assays when using the same concentration of the initiator template. CONCLUSIONS The Uni-Control method for preparing real-time PCR positive reaction controls provides an interim measure by which real-time PCR assays can be rapidly introduced for rare or emerging viruses in the absence of wild-type control material. The system is versatile and we propose can readily be adapted to almost any viral template.

Influenza Type C

Serological and molecular methods indicate influenza C virus is globally distributed and that the majority of the population has antibodies to the virus [1–3]. This method is a real time RT-PCR duplex assay targeting the matrix protein and non-structural genes of influenza C virus.

A Novel Duplex Real-Time Reverse-Transcription PCR Assay for the Detection of Influenza A and the Novel Influenza A(H1N1) Strain

Timely implementation of antiviral treatment and other public health based responses are dependent on accurate and rapid diagnosis of the novel pandemic influenza A(H1N1) strain. In this study we developed a duplex real-time PCR (RT-PCR) (dFLU-TM) assay for the simultaneous detection of a broad range of influenza A subtypes and specific detection of the novel H1N1 2009 pandemic strain. The assay was compared to the combined results of two previously described monoplex RT-PCR assays using 183 clinical samples and 10 seasonal influenza A isolates. Overall, the results showed that the dFLU-TM RT-PCR method is suitable for detection of influenza A, including the novel H1N1 pandemic strain, in clinical samples.