Viral infectivity in paediatric SARS-CoV-2 clinical samples does not vary by age.

At the start of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there was much uncertainty about the role of children in infection and transmission dynamics. Through the course of the pandemic, it became clear that children were susceptible to SARS-CoV-2 infection, although they were experiencing a notable lack of severe disease outcomes as compared to the adult population. This trend held true with the emergence of new SARS-CoV-2 variants, even in paediatric populations that were ineligible to be vaccinated. The difference in disease outcomes has prompted questions about the virological features of SARS-CoV-2 infection in this population. In order to determine if there was any difference in the infectivity of the virus produced by children with coronavirus disease 2019 (COVID-19), we compared viral RNA levels (clinical RT-qPCR C T) and infectious virus titres from 144 SARS-CoV-2-positive clinical samples collected from children aged 0 to 18 years old. We found that age had no impact on the infectiousness of SARS-CoV-2 within our cohort, with children of all ages able to produce high levels of infectious virus.

Viral infectivity in pediatric SARS-CoV-2 clinical samples does not vary by age.

During the early months of the SARS-CoV-2 pandemic, notable uncertainty emerged regarding the role of children in transmission dynamics. With time, it became more clear that children were susceptible to infection with SARS-CoV-2, but that the vast majority of children experienced mild symptoms with lower incidence of severe disease. This pattern remained consistent despite the later emergence of SARS-CoV-2 variants, including Delta and Omicron, even among children <5 ineligible for vaccination. The relative lack of severe disease in the pediatric population raised questions regarding viral kinetics and infectivity in children versus adults. We hypothesized that unique virologic features in children could explain this apparent decrease in symptoms and transmissibility early in the pandemic.

Sequencing and analysis of globally obtained human parainfluenza viruses 1 and 3 genomes.

Human Parainfluenza viruses (HPIV) type 1 and 3 are important causes of respiratory tract infections in young children globally. HPIV infections do not confer complete protective immunity so reinfections occur throughout life. Since no effective vaccine is available for the two virus subtypes, comprehensive understanding of HPIV-1 and HPIV-3 genetic and epidemic features is important for diagnosis, prevention, and treatment of HPIV-1 and HPIV-3 infections. Relatively few whole genome sequences are available for both HPIV-1 and HPIV-3 viruses, so our study sought to provide whole genome sequences from multiple countries to further the understanding of the global diversity of HPIV at a whole-genome level. We collected HPIV-1 and HPIV-3 samples and isolates from Argentina, Australia, France, Mexico, South Africa, Switzerland, and USA from the years 2003-2011 and sequenced the genomes of 40 HPIV-1 and 75 HPIV-3 viruses with Sanger and next-generation sequencing with the Ion Torrent, Illumina, and 454 platforms. Phylogenetic analysis showed that the HPIV-1 genome is evolving at an estimated rate of 4.97 × 10-4 mutations/site/year (95% highest posterior density 4.55 × 10-4 to 5.38 × 10-4) and the HPIV-3 genome is evolving at a similar rate (3.59 × 10-4 mutations/site/year, 95% highest posterior density 3.26 × 10-4 to 3.94 × 10-4). There were multiple genetically distinct lineages of both HPIV-1 and 3 circulating on a global scale. Further surveillance and whole-genome sequencing are greatly needed to better understand the spatial dynamics of these important respiratory viruses in humans.

Establishment of a lethal aged mouse model of human respiratory syncytial virus infection.

Human respiratory syncytial virus (HRSV) infection is a significant cause of morbidity and mortality, particularly among the children and the elderly. Despite extensive efforts, there is currently no formally approved vaccine and effective antiviral options against HRSV infection are limited. The development of vaccines and antiviral strategies for HRSV was partly hampered by the lack of an efficient lethal mouse model to evaluate the efficacy of the candidate vaccines or antivirals. In this study, we established a lethal HRSV mouse model by consecutively passaging a clinical HRSV isolate, GZ08-0. GZ08-18 was isolated from mouse bronchioalveolar lavage fluids at the 50th passage of GZ08-0. Importantly, all GZ08-18-inoculated mice succumbed to the infection by day 7 post infection, whereas all GZ08-0-inoculated mice recovered from the infection. Subsequent investigations demonstrated that GZ08-18 replicated to a higher titer in mouse lungs, induced more prominent lung pathology, and resulted in higher expression levels of a number of key pro-inflammatory cytokines including IFN-γ, MIP-1α, and TNF-α in comparison to that of GZ08-0. The cyclophosphamide pretreatment rendered the mice more susceptible to a lethal outcome with less rounds of virus inoculation. Full genome sequencing revealed 17 mutations in GZ08-18, some of which might account for the dramatically increased pathogenicity over GZ08-0. In addition, by using ribavirin as a positive control, we demonstrated the potential application of this lethal mouse model as a tool in HRSV investigations. Overall, we have successfully established a practical lethal mouse model for HRSV with a mouse-adapted virus, which may facilitate future in vivo studies on the evaluation of candidate vaccines and drugs against HRSV.

Simulated Respiratory Secretion for Use in the Development of Influenza Diagnostic Assays.

Many assays have been developed for the detection of influenza virus which is an important respiratory pathogen. Development of these assays commonly involves the use of human clinical samples for validation of their performance. However, clinical samples can be difficult to obtain, deteriorate over time, and be inconsistent in composition. The goal of this study was to develop a simulated respiratory secretion (SRS) that could act as a surrogate for clinical samples. To this end, we determined the effects major respiratory secretion components (Na+, K+, Ca2+, cells, albumin IgG, IgM, and mucin) have on the performance of influenza assays including both nucleic acid amplification and rapid antigen assays. Minimal effects on the molecular assays were observed for all of the components tested, except for serum derived human IgG, which suppressed the signal of the rapid antigen assays. Using dot blots we were able to show anti-influenza nucleoprotein IgG antibodies are common in human respiratory samples. We composed a SRS that contained mid-point levels of human respiratory sample components and studied its effect compared to phosphate buffered saline and virus negative clinical sample matrix on the Veritor, Sofia, CDC RT-PCR, Simplexa, cobas Liat, and Alere i influenza assays. Our results demonstrated that a SRS can interact with a variety of test methods in a similar manner to clinical samples with a similar impact on test performance.

Complete genome sequences of one human respiratory syncytial antigenic group a virus from china and its four mouse-adapted isolates.

In this study, one human respiratory syncytial antigenic group A virus (HRSV-A-GZ08-0) and its four BALB/c mouse-adapted isolates were sequenced and elucidated. Nineteen nucleotides were mutated between HRSV-A-GZ08-0 and the four mouse-adapted isolates.

Sequencing and analysis of globally obtained human respiratory syncytial virus A and B genomes.

BACKGROUND: Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in children globally, with nearly all children experiencing at least one infection by the age of two. Partial sequencing of the attachment glycoprotein gene is conducted routinely for genotyping, but relatively few whole genome sequences are available for RSV. The goal of our study was to sequence the genomes of RSV strains collected from multiple countries to further understand the global diversity of RSV at a whole-genome level. METHODS: We collected RSV samples and isolates from Mexico, Argentina, Belgium, Italy, Germany, Australia, South Africa, and the USA from the years 1998-2010. Both Sanger and next-generation sequencing with the Illumina and 454 platforms were used to sequence the whole genomes of RSV A and B. Phylogenetic analyses were performed using the Bayesian and maximum likelihood methods of phylogenetic inference. RESULTS: We sequenced the genomes of 34 RSVA and 23 RSVB viruses. Phylogenetic analysis showed that the RSVA genome evolves at an estimated rate of 6.72 × 10(-4) substitutions/site/year (95% HPD 5.61 × 10(-4) to 7.6 × 10(-4)) and for RSVB the evolutionary rate was 7.69 × 10(-4) substitutions/site/year (95% HPD 6.81 × 10(-4) to 8.62 × 10(-4)). We found multiple clades co-circulating globally for both RSV A and B. The predominant clades were GA2 and GA5 for RSVA and BA for RSVB. CONCLUSIONS: Our analyses showed that RSV circulates on a global scale with the same predominant clades of viruses being found in countries around the world. However, the distribution of clades can change rapidly as new strains emerge. We did not observe a strong spatial structure in our trees, with the same three main clades of RSV co-circulating globally, suggesting that the evolution of RSV is not strongly regionalized.

Analytical reactivity of 13 commercially available rapid influenza diagnostic tests with H3N2v and recently circulating influenza viruses.

OBJECTIVES: Rapid influenza diagnostic tests (RIDTs) used widely in clinical practice are simple to use and provide results within 15 minutes; however, reported performance is variable, which causes concern when novel or variant viruses emerge. This study's goal was to assess the analytical reactivity of 13 RIDTs with recently circulating seasonal and H3N2v influenza viruses, using three different viral measures. DESIGN: Virus stocks were characterized by infectious dose (ID50 ) and nucleoprotein (NP) concentration, diluted at half-log dilutions, and tested with each RIDT and real-time RT-PCR. RESULTS: Strong correlation was observed between NP concentration and RIDT reactivity; however, only weak correlation was seen with ID50 or Ct values. Only four RIDTs detected viral NP at the lowest dilution for all influenza A viruses (IAV). Influenza A viruses not detected by more than one RIDT had lower NP levels. Of the 13 RIDTs, 9 had no significant differences in reactivity across IAV when compared to NP levels. CONCLUSIONS: Previous reports of RIDT performance typically compare reactivity based on ID50 titers, which in this study correlated only weakly with proportional amounts of viral NP in prepared virus samples. In the context of the strong correlation of RIDT reactivity with NP concentration, H3N2v was found to be as reactive as seasonal circulating IAV. While these findings may not reflect clinical performance of these RIDTs, measuring NP concentration can be useful in the future to assess comparable reactivity of available RIDTs, or to assess reactivity with newly evolving or emerging viruses.

Genome sequencing and phylogenetic analysis of 39 human parainfluenza virus type 1 strains isolated from 1997-2010.

Thirty-nine human parainfluenza type 1 (HPIV-1) genomes were sequenced from samples collected in Milwaukee, Wisconsin from 1997-2010. Following sequencing, phylogenetic analyses of these sequences plus any publicly available HPIV-1 sequences (from GenBank) were performed. Phylogenetic analysis of the whole genomes, as well as individual genes, revealed that the current HPIV-1 viruses group into three different clades. Previous evolutionary studies of HPIV-1 in Milwaukee revealed that there were two genotypes of HPIV-1 co-circulating in 1991 (previously described as HPIV-1 genotypes C and D). The current study reveals that there are still two different HPIV-1 viruses co-circulating in Milwaukee; however, both groups of HPIV-1 viruses are derived from genotype C indicating that genotype D may no longer be in circulation in Milwaukee. Analyses of genetic diversity indicate that while most of the genome is under purifying selection some regions of the genome are more tolerant of mutation. In the 40 HPIV-1 genomes sequenced in this study, the nucleotide sequence of the L gene is the most conserved while the sequence of the P gene is the most variable. Over the entire protein coding region of the genome, 81 variable amino acid residues were observed and as with nucleotide diversity, the P protein seemed to be the most tolerant of mutation (and contains the greatest proportion of non-synonymous to synonymous substitutions) while the M protein appears to be the least tolerant of amino acid substitution.

Whole genome sequencing and evolutionary analysis of human respiratory syncytial virus A and B from Milwaukee, WI 1998-2010.

BACKGROUND: Respiratory Syncytial Virus (RSV) is the leading cause of lower respiratory-tract infections in infants and young children worldwide. Despite this, only six complete genome sequences of original strains have been previously published, the most recent of which dates back 35 and 26 years for RSV group A and group B respectively. METHODOLOGY/PRINCIPAL FINDINGS: We present a semi-automated sequencing method allowing for the sequencing of four RSV whole genomes simultaneously. We were able to sequence the complete coding sequences of 13 RSV A and 4 RSV B strains from Milwaukee collected from 1998-2010. Another 12 RSV A and 5 RSV B strains sequenced in this study cover the majority of the genome. All RSV A and RSV B sequences were analyzed by neighbor-joining, maximum parsimony and Bayesian phylogeny methods. Genetic diversity was high among RSV A viruses in Milwaukee including the circulation of multiple genotypes (GA1, GA2, GA5, GA7) with GA2 persisting throughout the 13 years of the study. However, RSV B genomes showed little variation with all belonging to the BA genotype. For RSV A, the same evolutionary patterns and clades were seen consistently across the whole genome including all intergenic, coding, and non-coding regions sequences. CONCLUSIONS/SIGNIFICANCE: The sequencing strategy presented in this work allows for RSV A and B genomes to be sequenced simultaneously in two working days and with a low cost. We have significantly increased the amount of genomic data that is available for both RSV A and B, providing the basic molecular characteristics of RSV strains circulating in Milwaukee over the last 13 years. This information can be used for comparative analysis with strains circulating in other communities around the world which should also help with the development of new strategies for control of RSV, specifically vaccine development and improvement of RSV diagnostics.

Epidemiologic Observations from Passive and Targeted Surveillance during the First Wave of the 2009 H1N1 Influenza Pandemic in Milwaukee, WI.

The first wave of the 2009 influenza H1N1 pandemic (H1N1pdm) in Milwaukee, WI has been recognized as the largest reported regional outbreak in the United States. The epidemiologic and clinical characteristics of this large first wave outbreak from April 28(th) 2009-July 25(th) 2009, studied using both passive and targeted surveillance methodologies are presented. A total of 2791 individuals with H1N1pdm infection were identified; 60 % were 5-18 years old. The 5-18 year and 0-4 year age groups had high infection (1131 and 1101 per 100,000) and hospitalization (49 and 12 per 100,000) rates respectively. Non-Hispanic blacks and Hispanics had the highest hospitalization and infection rates. In targeted surveillance, infected patients had fever (78%), cough (80%), sore throat (38%), and vomiting or diarrhea (8%). The "influenza like illness" definition captured only 68 % of infected patients. Modeling estimates that 10.3 % of Milwaukee population was infected in the first wave and 59% were asymptomatic. The distinct epidemiologic profile of H1N1pdm infections observed in the study has direct implications for predicting the burden of infection and hospitalization in the next waves of H1N1pdm. Careful consideration of demographic predictors of infection and hospitalization with H1N1pdm will be important for effective preparedness for subsequent influenza seasons.

N-linked glycan at residue 523 of human parainfluenza virus type 3 hemagglutinin-neuraminidase masks a second receptor-binding site.

The hemagglutinin-neuraminidase (HN) glycoprotein plays a critical role in parainfluenza virus replication. We recently found that in addition to the catalytic binding site, HN of human parainfluenza virus type 1 (hPIV-1) may have a second receptor-binding site covered by an N-linked glycan at residue 173, which is near the region of the second receptor-binding site identified in Newcastle disease virus (NDV) HN (I. A. Alymova, G. Taylor, V. P. Mishin, M. Watanabe, K. G. Murti, K. Boyd, P. Chand, Y. S. Babu, and A. Portner, J. Virol. 82:8400-8410, 2008). Sequence analysis and superposition of the NDV and hPIV-3 HN dimer structures revealed that, similar to what was seen in hPIV-1, the N-linked glycan at residue 523 on hPIV-3 HN may cover a second receptor-binding site. Removal of this N-linked glycosylation site by an Asn-to-Asp substitution at residue 523 (N523D) changed the spectrum of the mutant virus's receptor specificity, delayed its elution from both turkey and chicken red blood cells, reduced mutant sensitivity (by about half) to the selective HN inhibitor BCX 2855 in hemagglutination inhibition tests, and slowed its growth in LLC-MK(2) cells. The neuraminidase activity of the mutant and its sensitivity to BCX 2855 in neuraminidase inhibition assays did not change, indicating that the mutation did not affect the virus's catalytic-binding site and that all observed effects were caused by the exposure of the purported second receptor-binding site. Our data are consistent with the idea that, similar to the case for hPIV-1, the N-linked glycan shields a second receptor-binding site on hPIV-3 HN.

Development of a rapid automated influenza A, influenza B, and respiratory syncytial virus A/B multiplex real-time RT-PCR assay and its use during the 2009 H1N1 swine-origin influenza virus epidemic in Milwaukee, Wisconsin.

Rapid, semiautomated, and fully automated multiplex real-time RT-PCR assays were developed and validated for the detection of influenza (Flu) A, Flu B, and respiratory syncytial virus (RSV) from nasopharyngeal specimens. The assays can detect human H1N1, H3N2, and swine-origin (S-OIV) H1N1 Flu A viruses and were effectively used to distinguish Flu A infections (of all subtypes) from Flu B and RSV infections during the current S-OIV outbreak in Milwaukee, WI. The analytical limits of detection were 10(-2) to 10(1) TCID(50)/ml depending on the platform and analyte and showed only one minor cross-reaction among 23 common respiratory pathogens (intermittent cross-reaction to adenovirus at >10(7) TCID(50)/ml). A total of 100 clinical samples were tested by tissue culture, both automated assays, and the US Food and Drug Administration-approved ProFlu+ assay. Both the semiautomated and fully automated assays exhibited greater overall (Flu A, Flu B, and RSV combined) clinical sensitivities (93 and 96%, respectively) and individual Flu A sensitivities (100%) than the Food and Drug Administration-approved test (89% overall sensitivity and 93% Flu A sensitivity). All assays were 99% specific. During the S-OIV outbreak in Milwaukee, WI, the fully automated assay was used to test 1232 samples in 2 weeks. Flu A was detected in 134 clinical samples (126 H1N1 S-OIV, 5 H1N1 [human], and 1 untyped) with 100% positive agreement compared with other "in-house" validated molecular assays, with only 2 false-positive results. Such accurate testing using automated high-throughput molecule systems should allow clinicians and public health officials to react quickly and effectively during viral outbreaks.

The early diversification of influenza A/H1N1pdm.

Background Since its initial detection in April 2009, the A/H1N1pdm influenza virus has spread rapidly in humans, with over 5,700 human deaths. However, little is known about the evolutionary dynamics of H1N1pdm and its geographic and temporal diversification.Methods Phylogenetic analysis was conducted upon the concatenated coding regions of whole-genome sequences from 290 H1N1pdm isolates sampled globally between April 1 - July 9, 2009, including relatively large samples from the US states of Wisconsin and New York. Results At least 7 phylogenetically distinct viral clades have disseminated globally and co-circulated in localities that experienced multiple introductions of H1N1pdm. The epidemics in New York and Wisconsin were dominated by two different clades, both phylogenetically distinct from the viruses first identified in California and Mexico, suggesting an important role for founder effects in determining local viral population structures. Conclusions Determining the global diversity of H1N1pdm is central to understanding the evolution and spatial spread of the current pandemic, and to predict its future impact on human populations. Our results indicate that H1N1pdm has already diversified into distinct viral lineages with defined spatial patterns.

Rapid multiplex reverse transcription-PCR typing of influenza A and B virus, and subtyping of influenza A virus into H1, 2, 3, 5, 7, 9, N1 (human), N1 (animal), N2, and N7, including typing of novel swine origin influenza A (H1N1) virus, during the 2009 outbreak in Milwaukee, Wisconsin.

A large outbreak of novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) infection in Milwaukee, WI, occurred in late April 2009. We had recently developed a rapid multiplex reverse transcription-PCR enzyme hybridization assay (FluPlex) to determine the type (A or B) and subtype (H1, H2, H3, H5, H7, H9, N1 [human], N1 [animal], N2, or N7) of influenza viruses, and this assay was used to confirm the diagnoses for the first infected patients in the state. The analytical sensitivity was excellent at 1.5 to 116 copies/reaction, or 10(-3) to 10(-1) 50% tissue culture infective doses/ml. The testing of all existing hemagglutinin and neuraminidase subtypes of influenza A virus and influenza B virus (41 influenza virus strains) and 24 common respiratory pathogens showed only one low-level H3 cross-reaction with an H10N7 avian strain and only at 5.2 x 10(6) copies/reaction, not at lower concentrations. Comparisons of the FluPlex results with results from multiple validated in-house molecular assays, CDC-validated FDA-approved assays, and gene sequencing demonstrated 100% positive agreement for the typing of 179 influenza A viruses and 3 influenza B viruses, the subtyping of 110 H1N1 (S-OIV; N1 [animal]), 62 H1N1 (human), and 6 H3N2 (human) viruses, and the identification of 24 negative clinical samples and 100% negative agreement for all viruses tested except H1N1 (human) (97.7%). The small number of false-positive H1N1 (human) samples most likely represent increased sensitivity over that of other in-house assays, with four of four results confirmed by the CDC's influenza virus subtyping assay. The FluPlex is a rapid, inexpensive, sensitive, and specific method for the typing and subtyping of influenza viruses and demonstrated outstanding utility during the first 2 weeks of an S-OIV infection outbreak. Methods for rapid detection and broad subtyping of influenza viruses, including animal subtypes, are needed to address public concern over the emergence of pandemic strains. Attempts to automate this assay are ongoing.

Rapid semiautomated subtyping of influenza virus species during the 2009 swine origin influenza A H1N1 virus epidemic in Milwaukee, Wisconsin.

In the spring of 2009, a novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) emerged and began causing a large outbreak of illness in Milwaukee, WI. Our group at the Midwest Respiratory Virus Program laboratory developed a semiautomated real-time multiplex reverse transcription-PCR assay (Seasonal), employing the NucliSENS easyMAG system (bioMérieux, Durham, NC) and a Raider thermocycler (HandyLab Inc., Ann Arbor, MI), that typed influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) and subtyped influenza A virus into the currently circulating H1 and H3 subtypes, as well as a similar assay that identified H1 of S-OIV. The Seasonal and H1 S-OIV assays demonstrated analytical limits of detection of <50 50% tissue culture infective doses/ml and 3 to 30 input copies, respectively. Testing of the analytical specificities revealed no cross-reactivity with 41 and 26 different common organisms and demonstrated outstanding reproducibility of results. Clinical testing showed 95% sensitivity for influenza A virus and influenza B virus and 95 and 97% specificity compared to tissue culture. Comparisons of results from other molecular tests showed levels of positive agreement with the Seasonal and H1 S-OIV assay results of 99 and 100% and levels of negative agreement of 98 and 100%. This study has demonstrated the use of a semiautomated system for sensitive, specific, and rapid detection of influenza A virus, influenza B virus, and RSV and subtyping of influenza A virus into human H1 and H3 and S-OIV strains. This assay/system performed well in clinical testing of regular seasonal influenza virus subtypes and was outstanding during the 2009 Milwaukee S-OIV infection outbreak. This recent outbreak of infection with a novel influenza A (H1N1) virus also demonstrates the importance of quickly distributing information on new agents and of having rapid influenza virus subtyping assays widely available for clinical and public health decisions.

Multiplex assay for simultaneously typing and subtyping influenza viruses by use of an electronic microarray.

We report on the use of an electronic microarray to simultaneously type influenza A and B viruses and to distinguish influenza A virus subtypes H1N1 and H3N2 from the potentially pandemic avian virus subtype H5N1. The assay targets seven genes: the H1, H3, H5, N1, and N2 genes of influenza A virus; the matrix protein M1 gene of influenza A virus; and the nonstructural protein (NS) gene of influenza B virus. By combining a two-step reverse transcription-multiplex PCR with typing and subtyping on the electronic microarray, the assay achieved an analytical sensitivity of 10(2) to 10(3) copies of transcripts per reaction for each of the genes. The assay correctly typed and subtyped 15 different influenza virus isolates, including two influenza B virus, five A/H1N1, six A/H3N2, and two A/H5N1 isolates. In addition, the assay correctly identified 8 out of 10 diluted, archived avian influenza virus specimens with complete typing and subtyping information and 2 specimens with partial subtyping information. In a study of 146 human clinical specimens that had previously been shown to be positive for influenza virus or another respiratory virus, the assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The assay is a rapid, accurate, user-friendly method for simultaneously typing and subtyping influenza viruses.

Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays.

Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA) were developed to simultaneously detect many of the CDC category "A" bioterrorism agents. The "Bio T" DNA assay was developed to detect: Variola major (VM), Bacillus anthracis (BA), Yersinia pestis (YP), Francisella tularensis (FT) and Varicella zoster virus (VZV). The "Bio T" RNA assay (mRT-PCR-EHA) was developed to detect: Ebola virus (Ebola), Lassa fever virus (Lassa), Rift Valley fever (RVF), Hantavirus Sin Nombre species (HSN) and dengue virus (serotypes 1-4). Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked) clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD)) of the DNA asssay for genomic DNA was 1x10(0)~1x10(2) copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1x10(-2) TCID(50)/mL. The LOD for recombinant controls ranged from 1x10(2)~1x10(3)copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1x10(4)~1x10(6) copies/mL without extraction and 1x10(5)~1x10(6) copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1x10(-4) dilution for dengue 1 and 2, 1x10(4) LD(50)/mL and 1x10(2) LD(50)/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1x10(3) copies/mL (1.5 input copies/reaction) for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The surrogate sensitivities of these two assays were 100% (95%CI 83-100) for FT, BA (pX02), YP, VM, VZV, dengue 2,3,4 and 95% (95%CI 75-100) for BA (pX01) and dengue 1 using spiked clinical specimens. The specificity of both BioT multiplex assays on spiked specimens was 100% (95% CI 99-100). Compared to other available assays (culture, serology, PCR, etc.) both the BioT DNA mPCR-EHA and BioT RNA mRT-PCR-EHA are rapid, sensitive and specific assays for detecting many category "A" Bioterrorism agents using a standard thermocycler.

Detection of 11 common viral and bacterial pathogens causing community-acquired pneumonia or sepsis in asymptomatic patients by using a multiplex reverse transcription-PCR assay with manual (enzyme hybridization) or automated (electronic microarray) detection.

Community-acquired pneumonia (CAP) and sepsis are important causes of morbidity and mortality. We describe the development of two molecular assays for the detection of 11 common viral and bacterial agents of CAP and sepsis: influenza virus A, influenza virus B, respiratory syncytial virus A (RSV A), RSV B, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Legionella micdadei, Bordetella pertussis, Staphylococcus aureus, and Streptococcus pneumoniae. Further, we report the prevalence of carriage of these pathogens in respiratory, skin, and serum specimens from 243 asymptomatic children and adults. The detection of pathogens was done using both a manual enzyme hybridization assay and an automated electronic microarray following reverse transcription and PCR amplification. The analytical sensitivities ranged between 0.01 and 100 50% tissue culture infective doses, cells, or CFU per ml for both detection methods. Analytical specificity testing demonstrated no significant cross-reactivity among 19 other common respiratory organisms. One hundred spiked "surrogate" clinical specimens were all correctly identified with 100% specificity (95% confidence interval, 100%). Overall, 28 (21.7%) of 129 nasopharyngeal specimens, 11 of 100 skin specimens, and 2 of 100 serum specimens from asymptomatic subjects tested positive for one or more pathogens, with S. pneumoniae and S. aureus giving 89% of the positive results. Our data suggest that asymptomatic carriage makes the use of molecular assays problematic for the detection of S. pneumoniae or S. aureus in upper respiratory tract secretions; however, the specimens tested showed virtually no carriage of the other nine viral and bacterial pathogens, and the detection of these pathogens should not be a significant diagnostic problem. In addition, slightly less sensitive molecular assays may have better correlation with clinical disease in the case of CAP.

The Influenza Primer Design Resource: a new tool for translating influenza sequence data into effective diagnostics.

BACKGROUND: Recent outbreaks of highly pathogenic avian influenza and multiple occurrences of zoonotic infection and deaths in humans have sparked a dramatic increase in influenza research. In order to rapidly identify and help prevent future influenza outbreaks, numerous laboratories around the world are working to develop new nucleotide-based diagnostics for identifying and subtyping influenza viruses. While there are several databases that have been developed for manipulating the vast amount of influenza genetic data that have been produced, significant progress can still be made in developing tools for translating the genetic data into effective diagnostics. DESCRIPTION: The Influenza Primer Design Resource (IPDR) is the combination of a comprehensive database of influenza nucleotide sequences and a web interface that provides several important tools that aid in the development of oligonucleotides that may be used to develop better diagnostics. IPDR's database can be searched using a variety of criteria, allowing the user to align the subset of influenza sequences that they are interested in. In addition, IPDR reports a consensus sequence for the alignment along with sequence polymorphism information, a summary of most published primers and probes that match the consensus sequence, and a Primer3 analysis of potential primers and probes that could be used for amplifying the sequence subset. CONCLUSIONS: The IPDR is a unique combination of bioinformatics tools that will greatly aid researchers in translating influenza genetic data into diagnostics, which can effectively identify and subtype influenza strains. The website is freely available at http://www.ipdr.mcw.edu.