Selection of DNA aptamers that bind to influenza A viruses with high affinity and broad subtype specificity

AbstractMany cases of influenza are reported worldwide every year. The influenza virus often acquires new antigenicity, which is known as antigenic shift; this results in the emergence of new virus strains, for which preexisting immunity is not found in the population resulting in influenza pandemics. In the event a new strain emerges, diagnostic tools must be developed rapidly to detect the novel influenza strain. The generation of high affinity antibodies is costly and takes time; therefore, an alternative detection system, aptamer detection, provides a viable alternative to antibodies as a diagnostic tool. In this study, we developed DNA aptamers that bind to HA1 proteins of multiple influenza A virus subtypes by the SELEX procedure. To evaluate the binding properties of these aptamers using colorimetric methods, we developed a novel aptamer-based sandwich detection method employing our newly identified aptamers. This novel sandwich enzyme-linked aptamer assay successfully detected the H5N1, H1N1, and H3N2 subtypes of influenza A virus with almost equal sensitivities. These findings suggest that our aptamers are attractive candidates for use as simple and sensitive diagnostic tools that need sandwich system for detecting the influenza A virus with broad subtype specificities

H5N1 Influenza Viruses in Lao People’s Democratic Republic

A prospective surveillance program for influenza viruses was established in Lao People's Democratic Republic (PDR) in July of 2005. We report isolation of H5N1 virus genetically distinct from H5N1 circulating in 2004, which indicates reintroduction of H5N1 into Lao PDR after its disappearance (i.e., no virologic or serologic evidence) for 2 years

Oseltamivir–Resistant Pandemic H1N1/2009 Influenza Virus Possesses Lower Transmissibility and Fitness in Ferrets

The neuraminidase (NA) inhibitor oseltamivir offers an important immediate option for the control of influenza, and its clinical use has increased substantially during the recent H1N1 pandemic. In view of the high prevalence of oseltamivir-resistant seasonal H1N1 influenza viruses in 2007–2008, there is an urgent need to characterize the transmissibility and fitness of oseltamivir-resistant H1N1/2009 viruses, although resistant variants have been isolated at a low rate. Here we studied the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, one oseltamivir-sensitive and one resistant, in the ferret model. The resistant H275Y mutant was derived from a patient on oseltamivir prophylaxis and was the first oseltamivir-resistant isolate of the pandemic virus. Full genome sequencing revealed that the pair of viruses differed only at NA amino acid position 275. We found that the oseltamivir-resistant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets (0/2), while it retained efficient transmission via direct contact (2/2). The sensitive H1N1/2009 virus was efficiently transmitted via both routes (2/2 and 1/2, respectively). The wild-type H1N1/2009 and the resistant mutant appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. We compared viral fitness within the host by co-infecting a ferret with oseltamivir-sensitive and -resistant H1N1/2009 viruses and found that the resistant virus showed less growth capability (fitness). The NA of the resistant virus showed reduced substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of possible evolving viral proteins during the pandemic

A Genome-Wide Gene Function Prediction Resource for Drosophila melanogaster

Predicting gene functions by integrating large-scale biological data remains a challenge for systems biology. Here we present a resource for Drosophila melanogaster gene function predictions. We trained function-specific classifiers to optimize the influence of different biological datasets for each functional category. Our model predicted GO terms and KEGG pathway memberships for Drosophila melanogaster genes with high accuracy, as affirmed by cross-validation, supporting literature evidence, and large-scale RNAi screens. The resulting resource of prioritized associations between Drosophila genes and their potential functions offers a guide for experimental investigations

Avian Infectious Bronchitis Virus: A Tropism for the Rooster Reproductive Tract and Effects on Reproductive Performance

136 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.The epididymal region of the rooster consists of the rete testes, the efferent ductules and the epididymal duct. In the epididymal region, the spermatozoa gain the ability to move and acquire surface proteins. The specialized microenvironment of the epididymal region is vital for these developments to occur. A dysfunction of the epididymal region, epididymal stones, has been shown to affect fertility in the rooster. The cause of these epididymal stones was investigated and avian infectious bronchitis virus (AIBV) has been identified as a cause. AIBV is a highly contagious respiratory disease in chickens. The pathogenesis of AIBV in the female reproductive tract has been well studied; however, there have been few studies concerning the effects of AIBV on the rooster reproductive tract. Therefore this investigation was undertaken to identify the role of AIBV in epididymal stone formation and to characterize changes in seminal plasma composition. Specific objectives were: (1) to determine if AIBV can infect the epididymal region; (2) to investigate if vaccination with killed AIBV would result in epididymal stone formation; (3) to determine if AIBV affects fertility by altering composition of seminal plasma; and (4) to characterize protein changes in seminal plasma in response to AIBV vaccination with two dimensional gel electrophoresis. There were four major findings; First, replication of AIBV in the epididymal region of the rooster reproductive tract was detected using 3' RACE indicating the epididymal region is a target for the virus in vitro. Second, vaccination with killed AIBV did not reduce the incidence of epididymal stones; therefore, entrance and replication of AIBV in the reproductive tract is not required for stone formation. Third, seminal plasma protein patterns and free amino acid levels are altered in vaccinated roosters when compared to non-vaccinated roosters. Fourth, differentially expressed seminal plasma proteins were identified in vaccinated roosters when compared to non-vaccinated roosters. These data provide further evidence that the proper function of the efferent ductules is necessary for normal fertility and exposure to AIBV disrupts fertility.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Avian Infectious Bronchitis Virus: A Tropism for the Rooster Reproductive Tract and Effects on Reproductive Performance

136 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.The epididymal region of the rooster consists of the rete testes, the efferent ductules and the epididymal duct. In the epididymal region, the spermatozoa gain the ability to move and acquire surface proteins. The specialized microenvironment of the epididymal region is vital for these developments to occur. A dysfunction of the epididymal region, epididymal stones, has been shown to affect fertility in the rooster. The cause of these epididymal stones was investigated and avian infectious bronchitis virus (AIBV) has been identified as a cause. AIBV is a highly contagious respiratory disease in chickens. The pathogenesis of AIBV in the female reproductive tract has been well studied; however, there have been few studies concerning the effects of AIBV on the rooster reproductive tract. Therefore this investigation was undertaken to identify the role of AIBV in epididymal stone formation and to characterize changes in seminal plasma composition. Specific objectives were: (1) to determine if AIBV can infect the epididymal region; (2) to investigate if vaccination with killed AIBV would result in epididymal stone formation; (3) to determine if AIBV affects fertility by altering composition of seminal plasma; and (4) to characterize protein changes in seminal plasma in response to AIBV vaccination with two dimensional gel electrophoresis. There were four major findings; First, replication of AIBV in the epididymal region of the rooster reproductive tract was detected using 3' RACE indicating the epididymal region is a target for the virus in vitro. Second, vaccination with killed AIBV did not reduce the incidence of epididymal stones; therefore, entrance and replication of AIBV in the reproductive tract is not required for stone formation. Third, seminal plasma protein patterns and free amino acid levels are altered in vaccinated roosters when compared to non-vaccinated roosters. Fourth, differentially expressed seminal plasma proteins were identified in vaccinated roosters when compared to non-vaccinated roosters. These data provide further evidence that the proper function of the efferent ductules is necessary for normal fertility and exposure to AIBV disrupts fertility.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Efficacy of Oseltamivir Therapy in Ferrets Inoculated with Different Clades of H5N1 Influenza Virus

Highly pathogenic H5N1 influenza viruses have infected an increasing number of humans in Asia, with high mortality rates and the emergence of multiple distinguishable clades. It is not known whether antiviral drugs that are effective against contemporary human influenza viruses will be effective against systemically replicating viruses, such as these pathogens. Therefore, we evaluated the use of the neuraminidase (NA) inhibitor oseltamivir for early postexposure prophylaxis and for treatment in ferrets exposed to representatives of two clades of H5N1 virus with markedly different pathogenicities in ferrets. Ferrets were protected from lethal infection with the A/Vietnam/1203/04 (H5N1) virus by oseltamivir (5 mg/kg of body weight/day) given 4 h after virus inoculation, but higher daily doses (25 mg/kg) were required for treatment when it was initiated 24 h after virus inoculation. For the treatment of ferrets inoculated with the less pathogenic A/Turkey/15/06 (H5N1) virus, 10 mg/kg/day of oseltamivir was sufficient to reduce the lethargy of the animals, significantly inhibit inflammation in the upper respiratory tract, and block virus spread to the internal organs. Importantly, all ferrets that survived the initial infection were rechallenged with homologous virus after 21 days and were completely protected from infection. Direct sequencing of the NA or HA1 gene segments in viruses isolated from ferret after treatment showed no amino acid substitutions known to cause drug resistance in conserved residues. Thus, early oseltamivir treatment is crucial for protection against highly pathogenic H5N1 viruses and the higher dose may be needed for the treatment of more virulent viruses

Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis▿§

Neuraminidase (NA) inhibitors are among the first line of defense against influenza virus infection. With the increased worldwide use of the drugs, antiviral susceptibility surveillance is increasingly important for effective clinical management and for public health epidemiology. Effective monitoring requires effective resistance detection methods. We have developed and validated a novel genotyping method for rapid detection of established NA inhibitor resistance markers in influenza viruses by single nucleotide polymorphism (SNP) analysis. The multi- or monoplex SNP analysis based on single nucleotide extension assays was developed to detect NA mutations H275Y and I223R/V in pandemic H1N1 viruses, H275Y in seasonal H1N1 viruses, E119V and R292K in seasonal H3N2 viruses, and H275Y and N295S in H5N1 viruses. The SNP analysis demonstrated high sensitivity for low-content NA amplicons (0.1 to 1 ng/μl) and showed 100% accordant results against a panel of defined clinical isolates. The monoplex assays for the H275Y NA mutation allowed precise and accurate quantification of the proportions of wild-type and mutant genotypes in virus mixtures (5% to 10% discrimination), with results comparable to those of pyrosequencing. The SNP analysis revealed the lower growth fitness of an H275Y mutant compared to the wild-type pandemic H1N1 virus by quantitatively genotyping progeny viruses grown in normal human bronchial epithelial cells. This novel method offers high-throughput screening capacity, relatively low costs, and the wide availability of the necessary equipment, and thus it could provide a much-needed approach for genotypic screening of NA inhibitor resistance in influenza viruses