Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

BACKGROUND Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample. RESULTS EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 10² to 1.3 × 10⁸ copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10³ to 2.0 × 10⁵ copies/ml in infectious mononucleosis (n = 7), 7.5 × 10⁴ to 1.1 × 10⁵ copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10² to 5.6 × 10³ copies/ml in HIV-infected patients (n = 12), and 2.0 × 10² to 9.1 × 10⁴ copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11). CONCLUSION Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.The Ausimmune Study is funded by the National Multiple Sclerosis Society of the USA, the National Health & Medical Research Council (Project Grant 316901) and Multiple Sclerosis Research Australia

Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

<p>Abstract</p> <p>Background</p> <p>Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample.</p> <p>Results</p> <p>EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 10²to 1.3 × 10⁸copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10³to 2.0 × 10⁵copies/ml in infectious mononucleosis (n = 7), 7.5 × 10⁴to 1.1 × 10⁵copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10²to 5.6 × 10³copies/ml in HIV-infected patients (n = 12), and 2.0 × 10²to 9.1 × 10⁴copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11).</p> <p>Conclusion</p> <p>Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.</p

Molecular Identification and Analysis of Nonserotypeable Human Enteroviruses▿ †

Conventional approaches to characterizing human enteroviruses (HEVs) are based on viral isolation and neutralization. Molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) and nucleotide sequencing of the entire or partial VP1 gene. A modified RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient way to characterize common and nonserotypeable (by neutralization) HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at a reference laboratory from 1979 to 2007; these common serotypes were identified using one sense and three antisense primers and a set of 80 serotype-specific probes in VP1 (F. Zhou et al., J. Clin. Microbiol. 47:2737-2743, 2009). In this study, one HEV-specific primer pair, two probes in the 5′ untranslated region (UTR), and a new set of 80 serotype-specific probes in VP1 were designed. First, we successfully applied the modified RT-PCR-RLB (using two HEV-specific probes and two sets of serotype-specific probes) to synchronously detect the 5′ UTR and VP1 regions of 131/132 isolates previously studied (F. Zhou et al., J. Clin. Microbiol. 47:2737-2743, 2009). Then, this method was used to identify 73/92 nonserotypeable HEV isolates; 19 nonserotypeable isolates were hybridized only with HEV-specific probes. The VP1 region of 92 nonserotypeable HEV isolates was sequenced; 73 sequences corresponded with one or both RLB results and 19 (not belonging to the 20 most common genotypes) were identified only by sequencing. Two sets of serotype-specific probes can capture the majority of strains belonging to the 20 most common serotypes/genotypes simultaneously or complementarily. Synchronous detection of the 5′ UTR and VP1 region by RT-PCR-RLB will facilitate the identification of HEVs, especially nonserotypeable isolates

Outbreak of human metapneumovirus inflection in a residential aged care facility

Summer outbreaks of respiratory illness in residential aged care facilities are uncommonly reported in New South Wales. A respiratory illness outbreak in an aged care facility during January 2008 prompted a response to contain the outbreak by implementing infection control measures, including cohorting of symptomatic residents, cohorting nursing care, closure to new admissions and the use of personal protective equipment by staff. In addition, respiratory tract specimens were collected to determine the causative agent. Human metapneumovirus (hMPV) was detected by polymerase chain reaction assay in 3 specimens with no other respiratory pathogens found. This is the 1st reported outbreak of hMPV in an aged care facility in Australia. hPMV should be considered as the possible cause of outbreaks in aged care facilities when influenza and respiratory syncytial virus have been excluded

Nosocomial and community transmission of measles virus Genotype D8 imported by a returning traveller from Nepal

Measles is uncommon in Australia due to effective national vaccination strategies. In mid-2003, a cluster of nine cases of measles occurred in western Sydney. The index case was a 29-year-old traveller recently returned from Nepal. The case presented to hospital and transmitted the disease to two others in the Emergency Department. Further cases resulted from both community and nosocomial transmission. The median age of cases was 24 years, with three cases in children aged under four years. Only one person had a documented history of measles vaccination, a child who had received one dose of vaccine overseas. One case was a 2-month-old infant whose mother was immune and two cases were hospital staff members. Molecular analysis of measles virus isolates from four cases revealed the same D8 genotype, a strain previously identifi ed in Nepal. Staff vaccination strategies implemented as a result of the outbreak were poorly patronised despite nosocomial transmission. As diseases such as measles become rare it is important to thoroughly investigate any outbreaks, and to maintain a high index of suspicion of measles, particularly in travellers presenting with a rash having returned from measles-endemic areas. Genetic analysis is important in tracing the origins of an outbreak, and to confi rm relatedness between cases. The highly infectious nature of measles virus also underscores the need for appropriate infection control in minimising risk of nosocomial transmission. Such policies are of increasing importance with the emergence of novel viruses or the threat of pandemic infl uenza

Epstein-Barr virus genotypes and strains in central nervous system demyelinating disease and Epstein-Barr virus-related illnesses in Australia

Objectives: To identify Epstein-Barr virus (EBV) genotypes and strains in samples from individuals with and without a first diagnosis of central nervous system (CNS) demyelinating disease (a possible precursor to multiple sclerosis) and patients with EBV-associated diseases in Australia. Methods: Samples from 55 EBV DNA and serology positive subjects including individuals with (n = 17) and without (n = 21) a first clinical diagnosis of CNS demyelination and patients with EBV-related diseases (n = 17) were examined. EBV genotype and strain were identified by sequence mutations within the Epstein-Barr nuclear antigen-2 region (EBNA-2) using DNA sequence analysis. Results: Both EBV genotypes, A and B, were detected (genotype A, 54/55, 98.2%; genotype B, 1/55, 1.8%). Within genotype A, GD1 was the most commonly detected strain (42/54, 77.7%), followed by B95-8 (9/54, 16.7%) and M-ABA (3/54, 5.6%). Genotype B, strain AG876, was found in one individual with CNS demyelinating disease. Conclusions: EBV genotype A and the GD1 strain were the common EBV genotypes isolated from individuals with and without CNS demyelinating disease, and in subjects with various EBV-related diseases. Although disease-specific genotypes or strains were not identified, this study provides useful insights into the molecular epidemiology of EBV infection in Australia

SHORT REPORT Open Access

Pandemic clinical case definitions are nonspecific: multiple respiratory viruses circulating in the early phases of the 2009 influenza pandemi

Identification of 20 Common Human Enterovirus Serotypes by Use of a Reverse Transcription-PCR-Based Reverse Line Blot Hybridization Assay ▿

The more than 100 human enterovirus (HEV) serotypes can also be classified into four species, HEV-A to -D, based on phylogenetic analysis of multiple gene regions. Current molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) amplification and nucleotide sequencing of the entire or 3′ half of the VP1 gene. An RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient approach to characterize common HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at an Australian reference virology laboratory from 1979 to 2007. VP1 sequences of all known HEV prototype strains were aligned to design one sense primer and three antisense primers for RT-PCR. After sequencing of the complete VP1 genes of 37 previously serotyped examples of the commonest 20 serotypes and alignment of these VP1 sequences with GenBank sequences, four serotype-specific probes for each serotype were designed for RLB. The RT-PCR-RLB assay was then applied to 132 HEV isolates, made up of the previously sequenced 37 isolates and another 95 serotyped clinical isolates. The RT-PCR-RLB genotypes corresponded with the serotypes for 131/132 isolates; the one exception was confirmed by VP1 sequencing, and the genotype was confirmed by repeat conventional serotyping. Genotyping by RT-PCR-RLB complements traditional serotyping methods and VP1 sequencing and has the advantages of convenience, speed, and accuracy. RT-PCR-RLB allows detection of specific enteroviral serotypes or genotypes associated with HEV outbreaks and significant disease