Ian M. Mackay

Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections.

Viruses are the major cause of pediatric acute respiratory tract infection (ARTI) and yet many suspected cases of infection remain uncharacterized. We employed 17 PCR assays and retrospectively screened 315 specimens selected by season from a predominantly pediatric hospital‐based population. Before the Brisbane respiratory virus research study commenced, one or more predominantly viral pathogens had been detected in 15.2% (n = 48) of all specimens. The Brisbane study made an additional 206 viral detections, resulting in the identification of a microbe in 67.0% of specimens. After our study, the majority of microbes detected were RNA viruses (89.9%). Overall, human rhinoviruses (HRVs) were the most frequently identified target (n = 140) followed by human adenoviruses (HAdVs; n = 25), human metapneumovirus (HMPV; n = 18), human bocavirus (HBoV; n = 15), human respiratory syncytial virus (HRSV; n = 12), human coronaviruses (HCoVs; n = 11), and human herpesvirus‐6 (n = 11). HRVs were the sole microbe detected in 37.8% (n = 31) of patients with suspected lower respiratory tract infection (LRTI). Genotyping of the HRV VP4/VP2 region resulted in a proposed subdivision of HRV type A into sublineages A1 and A2. Most of the genotyped HAdV strains were found to be type C. This study describes the high microbial burden imposed by HRVs, HMPV, HRSV, HCoVs, and the newly identified virus, HBoV on a predominantly paediatric hospital population with suspected acute respiratory tract infections and proposes a new formulation of viral targets for future diagnostic research studies. J. Med. Virol. 78:1232–1240, 2006.

Evidence of human coronavirus HKU1 and human bocavirus in Australian children

Abstract Undiagnosed cases of respiratory tract disease suspected of an infectious aetiology peak during the winter months. Since studies applying molecular diagnostic assays usually report reductions in the number of undiagnosed cases of infectious disease compared to traditional techniques, we applied PCR assays to investigate the role of two recently described viruses, namely human coronavirus (HCoV) HKU1 and human bocavirus (HBoV), in a hospital-based paediatric population. Both viruses were found among Australia children with upper or lower respiratory tract disease during the autumn and winter of 2004, contributing to 21.1% of all microbial diagnoses, with individual incidences of 3.1% (HCoV-HKU1) and 5.6% (HBoV) among 324 specimens. HBoV was found to coincide with another virus in more than half of all instances and displayed a single genetic lineage, whilst HCoV-HKU1 was more likely to occur in the absence of another microbe and strains could be divided into two genetic lineages which we propose be termed HCoV-HKU1 type A and type B. Children under the age of 2 years were most at risk of infection by these viruses which contribute significantly to the microbial burden among patients with respiratory tract disease during the colder months.

Human Bocavirus: Passenger or Pathogen in Acute Respiratory Tract Infections?

SUMMARY Human bocavirus (HBoV) is a newly identified virus tentatively assigned to the family Parvoviridae, subfamily Parvovirinae, genus Bocavirus. HBoV was first described in 2005 and has since been detected in respiratory tract secretions worldwide. Herein we review the literature on HBoV and discuss the biology and potential clinical impact of this virus. Most studies have been PCR based and performed on patients with acute respiratory symptoms, from whom HBoV was detected in 2 to 19% of the samples. HBoV-positive samples have been derived mainly from infants and young children. HBoV DNA has also been detected in the blood of patients with respiratory tract infection and in fecal samples of patients with diarrhea with or without concomitant respiratory symptoms. A characteristic feature of HBoV studies is the high frequency of coinciding detections, or codetections, with other viruses. Available data nevertheless indicate a statistical association between HBoV and acute respiratory tract disease. We present a model incorporating these somewhat contradictory findings and suggest that primary HBoV infection causes respiratory tract symptoms which can be followed by prolonged low-level virus shedding in the respiratory tract. Detection of the virus in this phase will be facilitated by other infections, either simply via increased sample cell count or via reactivation of HBoV, leading to an increased detection frequency of HBoV during other virus infections. We conclude that the majority of available HBoV studies are limited by the sole use of PCR diagnostics on respiratory tract secretions, addressing virus prevalence but not disease association. The ability to detect primary infection through the development of improved diagnostic methods will be of great importance for future studies seeking to assign a role for HBoV in causing respiratory illnesses.

Characterisation of a newly identified human rhinovirus, HRV-QPM, discovered in infants with bronchiolitis

Abstract Background Human rhinoviruses (HRVs) are some of the earliest identified and most commonly detected viruses associated with acute respiratory tract infections (ARTIs) and yet the molecular epidemiology and genomic variation of individual serotypes remains undefined. Objectives To molecularly characterise a novel HRV and determine its prevalence and clinical impact on a predominantly paediatric population. Study design Nucleotide sequencing was employed to determine the complete HRV-QPM coding sequence. Two novel real-time RT-PCR diagnostic assays were designed and employed to retrospectively screen a well-defined population of 1244 specimen extracts to identify the prevalence of HRV-QPM during 2003. Results Phylogenetic studies of complete coding sequences defined HRV-QPM as a novel member the genus Rhinovirus residing within the previously described HRV-A2 sub-lineage. Investigation of the relatively short VP1 sequence suggest that the virus is resistant to Pleconaril, setting it apart from the HRV A species. Sixteen additional HRV-QPM strains were detected (1.4% of specimens) often as the sole micro-organism present among infants with suspected bronchiolitis. HRV-QPM was also detected in Europe during 2006, and a closely related virus circulated in the United States during 2004. Conclusions We present the molecular characterisation and preliminary clinical impact of a newly identified HRV along with sequences representing additional new HRVs.

Molecular Assays for Detection of Human Metapneumovirus

ABSTRACT The recent description of the respiratory pathogen human metapneumovirus (hMPV) has highlighted a deficiency in current diagnostic techniques for viral agents associated with acute lower respiratory tract infections. We describe two novel approaches to the detection of viral RNA by use of reverse transcriptase PCR (RT-PCR). The PCR products were identified after capture onto a solid-phase medium by hybridization with a sequence-specific, biotinylated oligonucleotide probe. The assay was applied to the screening of 329 nasopharyngeal aspirates sampled from patients suffering from respiratory tract disease. These samples were negative for other common microbial causes of respiratory tract disease. We were able to detect hMPV sequences in 32 (9.7%) samples collected from Australian patients during 2001. To further reduce result turnaround times we designed a fluorogenic TaqMan oligoprobe and combined it with the existing primers for use on the LightCycler platform. The real-time RT-PCR proved to be highly reproducible and detected hMPV in an additional 6 out of 62 samples (9.6%) tested during the comparison of the two diagnostic approaches. We found the real-time RT-PCR to be the test of choice for future investigation of samples for hMPV due to its speed, reproducibility, specificity, and sensitivity.

A Sensitive, Specific, and Cost-Effective Multiplex Reverse Transcriptase-PCR Assay for the Detection of Seven Common Respiratory Viruses in Respiratory Samples

Cell culture and direct fluorescent antibody (DFA) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections. Multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) is a sensitive, specific, and rapid method for detecting several DNA and RNA viruses in a single specimen. We developed a m-RT-PCR assay that utilizes multiple virus-specific primer pairs in a single reaction mix combined with an enzyme-linked amplicon hybridization assay (ELAHA) using virus-specific probes targeting unique gene sequences for each virus. Using this m-RT-PCR-ELAHA, we examined the presence of seven respiratory viruses in 598 nasopharyngeal aspirate (NPA) samples from patients with suspected respiratory infection. The specificity of each assay was 100%. The sensitivity of the DFA was 79.7% and the combined DFA/culture amplified-DFA (CA-DFA) was 88.6% when compared to the m-RT-PCR-ELAHA. Of the 598 NPA specimens screened by m-RT-PCR-ELAHA, 3% were positive for adenovirus (ADV), 2% for influenza A (Flu A) virus, 0.3% for influenza B (Flu B) virus, 1% for parainfluenza type 1 virus (PIV1), 1% for parainfluenza type 2 virus (PIV2), 5.5% for parainfluenza type 3 virus (PIV3), and 21% for respiratory syncytial virus (RSV). The enhanced sensitivity, specificity, rapid result turnaround time and reduced expense of the m-RT-PCR-ELAHA compared to DFA and CA-DFA, suggests that this assay would be a significant improvement over traditional assays for the detection of respiratory viruses in a clinical laboratory.

Real-Time PCR Assays for Detection of Bocavirus in Human Specimens

ABSTRACT The recently discovered human bocavirus (HBoV) is the first member of the family Parvoviridae, genus Bocavirus, to be potentially associated with human disease. Several studies have identified HBoV in respiratory specimens from children with acute respiratory disease, but the full spectrum of clinical disease and the epidemiology of HBoV infection remain unclear. The availability of rapid and reliable molecular diagnostics would therefore aid future studies of this novel virus. To address this, we developed two sensitive and specific real-time TaqMan PCR assays that target the HBoV NS1 and NP-1 genes. Both assays could reproducibly detect 10 copies of a recombinant DNA plasmid containing a partial region of the HBoV genome, with a dynamic range of 8 log units (101 to 108 copies). Eight blinded clinical specimen extracts positive for HBoV by an independent PCR assay were positive by both real-time assays. Among 1,178 NP swabs collected from hospitalized pneumonia patients in Sa Kaeo Province, Thailand, 53 (4.5%) were reproducibly positive for HBoV by one or both targets. Our data confirm the possible association of HBoV infection with pneumonia and demonstrate the utility of these real-time PCR assays for HBoV detection.

New human coronavirus, HCoV-NL63, associated with severe lower respiratory tract disease in Australia

A new human coronavirus, HCoV‐NL63, was associated recently with bronchiolitis. The current study aimed to examine retrospectively stored specimens for the presence of HCoV‐NL63 using nested RT‐PCR assays targeting the 1a and 1b genes. The study population was composed of patients with acute respiratory disease warranting presentation to Queensland hospitals. HCoV‐NL63 was detected in the nasopharyngeal aspirates (NPA) of 16 of 840 specimens representing 766 patients (2%). HCoV‐NL63 positive individuals were diagnosed most commonly with lower respiratory tract (LRT) disease (81%). The clinical diagnosis was commonly supported by an abnormal chest X‐ray (56%) together with respiratory distress (50%), wheeze (44%), and râles (25%) on first presentation with HCoV‐NL63 infection. All patients positive for HCoV‐NL63 required admission to hospital. Among 38% of HCoV‐NL63 positive specimens a second pathogen was detected. Sequencing of amplicon from gene 1b revealed more than 99% nucleotide homology with the viral type strains while sequencing amplicon from gene 1a permitted the grouping of viral strains. It was shown that HCoV‐NL63 is associated with severe LRT disease in an Australian hospital setting during the cooler months of the year. We propose that HCoV‐NL63 is a global and seasonal pathogen of both children and adults associated with severe LRT illness. J. Med. Virol. 75:455–462, 2005.

Global Genetic Diversity of Human Metapneumovirus Fusion Gene

We analyzed 64 human metapneumovirus strains from eight countries. Phylogenetic analysis identified two groups (A and B, amino acid identity 93%–96%) and four subgroups. Although group A strains predominated, accounting for 69% of all strains, as many B as A strains were found in persons >3 years of age.

Distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus C (HRV C).

Background Human rhinoviruses (HRVs) are the most frequently detected pathogens in acute respiratory tract infections (ARTIs) and yet little is known about the prevalence, recurrence, structure and clinical impact of individual members. During 2007, the complete coding sequences of six previously unknown and highly divergent HRV strains were reported. To catalogue the molecular and clinical features distinguishing the divergent HRV strains, we undertook, for the first time, in silico analyses of all available polyprotein sequences and performed retrospective reviews of the medical records of cases in which variants of the prototype strain, HRV-QPM, had been detected. Methodology/Principle Findings Genomic analyses revealed that the six divergent strains, residing within a clade we previously called HRV A2, had the shortest polyprotein of all picornaviruses investigated. Structure-based amino acid alignments identified conserved motifs shared among members of the genus Rhinovirus as well as substantive deletions and insertions unique to the divergent strains. Deletions mostly affected regions encoding proteins traditionally involved in antigenicity and serving as HRV and HEV receptor footprints. Because the HRV A2 strains cannot yet be cultured, we created homology models of predicted HRV-QPM structural proteins. In silico comparisons confirmed that HRV-QPM was most closely related to the major group HRVs. HRV-QPM was most frequently detected in infants with expiratory wheezing or persistent cough who had been admitted to hospital and required supplemental oxygen. It was the only virus detected in 65% of positive individuals. These observations contributed to an objective clinical impact ranging from mild to severe. Conclusions The divergent strains did not meet classification requirements for any existing species of the genus Rhinovirus or Enterovirus. HRV A2 strains should be partitioned into at least one new species, putatively called Human rhinovirus C, populated by members detected with high frequency, from individuals with respiratory symptoms requiring hospital admission.