Seweryn Bialasiewicz

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.

Presence of the newly discovered human polyomaviruses KI and WU in Australian patients with acute respiratory tract infection

Abstract Background Currently, the role of the novel human polyomaviruses, KI (KIV) and WU (WUV) as agents of human disease remains uncertain. Objectives We sought to determine the prevalence of these viruses and their rate of co-detection with other viral respiratory pathogens, in an Australian population. Study design Polymerase chain reaction assays previously described were used to examine the presence of KIV and WUV in 2866 respiratory specimens collected from January to December 2003 from Australian patients with acute respiratory infections. Results KIV and WUV were present in our population with an annual prevalence of 2.6% and 4.5%, respectively. There was no apparent seasonal variation for KIV, but a predominance of infection was detected during late winter to early summer for WUV. The level of co-infection of KIV or WUV with other respiratory viruses was 74.7% and 79.7%, respectively. Both viruses were absent from urine and blood specimens collected from a variety of patient sources. Conclusions KIV and WUV circulate annually in the Australian population. Although there is a strong association with the respiratory tract, more comprehensive studies are required to prove these viruses are agents causing respiratory disease.

Merkel Cell Polyomavirus DNA in Respiratory Specimens from Children and Adults

Merkel cell polyomavirus (MCPyV) DNA was detected in 7 (1.3%) of 526 respiratory tract samples from patients in Australia with upper or lower respiratory tract symptoms. Partial T antigen and major capsid protein sequences of MCPyV identified in respiratory secretions showed high homology (99%–100%) to those found in Merkel cell carcinoma.

Genetic Diversity of Human Metapneumovirus over 4 Consecutive Years in Australia

The molecular epidemiologic profile of human metapneumovirus (hMPV) infection has likely been skewed toward certain genetic subtypes because of assay-design issues, and no comprehensive studies have been conducted to date. Here, reverse-transcription polymerase chain reaction was used to screen 10,319 specimens from patients presenting to hospitals with suspected respiratory tract infections during 2001-2004. After analysis of 727 Australian hMPV strains, 640 were assigned to 1 of 4 previously described subtypes. hMPV was the most common pathogen detected, and subtype B1 was the most common lineage. Concurrent, annual circulation of all 4 hMPV subtypes in our study population was common, with a single, usually different hMPV subtype predominating in each year.

Detection of BK, JC, WU, or KI polyomaviruses in faecal, urine, blood, cerebrospinal fluid and respiratory samples

BACKGROUND The recently described WU (WUV) and KI (KIV) polyomaviruses have been primarily detected in respiratory samples, however other body sites have not been extensively investigated to date. The related human polyomaviruses JCV and BKV in contrast, have been detected in a wide range of sample types, leading to increased knowledge about their biology and pathogenesis. OBJECTIVES The aim of the study was to investigate and compare the presence of JCV, BKV, WUV, and KIV in a variety of patient samples. STUDY DESIGN Nasopharyngeal aspirates (NPAs), bronchoalveolar lavages (BALs), cerebrospinal fluid (CSF), blood, faeces and urine from paediatric and adult immunocompetent and compromised patients were screened for the presence of the polyomaviruses by real-time PCR. The non-translated region (NTR) and VP1 of select WUV and KIV positive samples were sequenced and analysed. RESULTS WUV and KIV were predominantly detected in NPA, BAL, and faeces from paediatric patients. JCV and BKV were primarily detected in blood, urine and faeces from adult patients. WUV and KIV NTR/VP1 sequence similarity ranged from 99.5% to 100% and 97.5-100%, respectively. CONCLUSIONS Overall, WUV and KIV were detected in paediatric respiratory tract samples, in contrast to JCV and BKV, in which respiratory detections were uncommon. Additionally, the lack of WUV and KIV detections in blood, CSF, urine and adult faeces reinforces the parallel in divergent genomic phylogeny and apparent tissue tropism between JCV and BKV, and WUV and KIV. NTR/VP1 sequence variation did not appear to be associated with site of WUV or KIV detection.

Use of the P Gene to Genotype Human Metapneumovirus Identifies 4 Viral Subtypes

This study, conducted during 2001-2003, undertook the screening of patients with acute infectious respiratory-tract disease. A random selection of positive specimens was used for sequencing studies of the human metapneumovirus (hMPV) nucleoprotein gene and the phosphoprotein (P) gene. Australian and international sequences were compared, and a global classification scheme was developed. The hMPV P gene was an ideal molecular target for the detection and genotyping of hMPV. The region contained conserved sequences for reverse-transcriptase-polymerase chain-reaction primers and adequate variability to permit the accurate genotyping of the virus into 2 main lineages and 4 sublineages. Establishing viral identity is essential for the linking of genotype and disease severity.

A newly reported human polyomavirus, KI virus, is present in the respiratory tract of Australian children

Abstract Background Recently, Allander and co-workers reported the discovery of a new human polyomavirus, KI virus, in respiratory secretions from patients with acute respiratory tract infection (ARTI). Objective We examined 951 respiratory samples collected in Queensland, Australia, between November 2002 and August 2003 from patients with respiratory infection, for the presence of the KI virus. Results Twenty-four (2.5%) samples were positive for KI virus with 20 (83%) of these from children younger than 5 years. In six (25%) patients KI was co-detected with another virus. Full genome sequencing of three isolates shows a high degree of conservation between the Queensland isolates and the original isolates reported from Swedish patients. Conclusions The newly described KI polyomavirus may commonly be found in the respiratory tract of patients with ARTI, particularly children, and results indicate that the virus has global presence.

Detection of human bocavirus in respiratory, fecal, and blood samples by real-time PCR.

Human bocavirus (HBoV) has been detected worldwide in respiratory samples. Two real‐time PCR assays, targeting the non‐structural protein (NP‐1) and viral protein (VP‐1) genes, were designed and validated to detect HBoV in patients with respiratory disease, gastroenteritis, or systemic illness. Sensitivity of the NP‐1 and VP‐1 assays were equal to the conventional PCR assay previously described by Allander et al. [2005: Proc Natl Acad Sci USA 102: 12891–12896] being 100%, and giving specificity of 94% and 93%, respectively. There was no cross‐reaction identified with unrelated respiratory agents, or to human DNA. The limits of detection were 10 copies of genomic DNA equivalents per reaction for both assays. The assays were used to screen three different sample populations, combined nose, and throat swabs (n = 96) from children with acute respiratory disease, fecal samples (n = 375) from adults, and children with gastroenteritis and whole blood (n = 229) collected from 31 immunocompromised children taken over an 18‐month period. In total 17 (18%) respiratory samples and 18 (4.8%) fecal samples were identified as having HBoV present. Of the pediatric whole blood specimens investigated, HBoV was detected in six (2.6%) samples from four patients. In summary, two real‐time PCR assays targeting different genes were designed and validated for use as screening methods for the detection of HBoV. HBoV was found in three different specimen types: parent‐collected combined nose–throat swabs, fecal samples collected from symptomatic individuals and whole blood from immunocompromised children. J. Med. Virol. 81:488–493, 2009.

Development and evaluation of real-time PCR assays for the detection of the newly identified KI and WU polyomaviruses.

Abstract Background Recently, novel human polyomaviruses, KI (KIV) and WU (WUV) were described. Their role in human disease has not yet been determined. Objectives The aim of this study was to develop sensitive and specific assays for the detection of KIV and WUV. Study Two KIV (KI-A and KI-B) and three WUV (WU-A, WU-B and WU-C) real-time polymerase chain reaction (rtPCR) assays were developed and evaluated. Clinical sensitivities and specificities were determined by testing 200 respiratory specimens and the results compared to those for previously described conventional PCR assays. Limits of detection were determined, and the analytical specificities of the assays were investigated. Results No cross-reactivity was observed between the rtPCR methods and unrelated organisms. All five rtPCR assays could reliably detect 10 copies of genomic DNA equivalents per reaction, which was more sensitive than conventional methods. Compared to the conventional PCR assays, the sensitivity of the KI-A, KI-B, WU-A, WU-B and WU-C assays was 100%, 86.7% 95.5%, 100% and 100%, respectively. Specificity was 94.6%, 97.3%, 96.6%, 97.7% and 97.2%, respectively. Conclusions The KI-A, WU-B and WU-C assays provide the most sensitive detection of KIV and WUV in clinical specimens and may be used for further research into these viruses.

Different serologic behavior of MCPyV, TSPyV, HPyV6, HPyV7 and HPyV9 polyomaviruses found on the skin.

The polyomavirus family is rapidly expanding with twelve new human viruses identified since 2007. A significant number of the new human polyomaviruses (HPyV) has been found on the skin. Whether these viruses share biological properties and should be grouped together is unknown. Here we investigated the serological behavior of cutaneous HPyVs in a general population. 799 sera from immunocompetent Australian individuals aged between 0–87 were analyzed with a Luminex xMAP technology-based immunoassay for the presence of VP1-directed IgG antibodies against MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, and BKPyV as a control. Except for HPyV9, overall seropositivity was high for the cutanous polyomaviruses (66–81% in adults), and gradually increased with age. Children below 6 months displayed seropositivity rates comparable to the adults, indicative of maternal antibodies. TSPyV seroreactivity levels strongly increased after age 2 and waned later in life comparable to BKPyV, whereas MCPyV, HPyV6 and HPyV7 seroreactivity remained rather stable throughout. Based on the identified serologic profiles, MCPyV seems to cluster with HPyV6 and HPyV7, and TSPyV and HPyV9 by themselves. These profiles indicate heterogeneity among cutaneous polyomaviruses and probably reflect differences in exposure and pathogenic behavior of these viruses.