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Immuno-Pathogenesis of Dengue Hemorrhagic Fever (DHF)

Prida Malasit, M.D.

Mahidol University

DHF is one of the major health problems in tropical and subtropical areas affecting and causing mortality and morbidity in predominantly children of school age. The disease is caused by dengue viruses of the Flavivirus genus, and transmitted by Aedes aegypti mosquito. Four serotypes of the viruses are usually found circulating in endemic areas. DHF has unique features that separate it from other flavivirus infections. First, DHF occurs almost exclusively in those who develop secondary immune response to a different heterotypic virus. In other words, virus infection does not per se cause DHF, but it is an ‚aberrant‛ immune response in a susceptible host, responding to a second but different serotype virus, that is responsible for the pathogenesis. Second, sudden onset of shock, plasma leakage and hemorrhagic diathesis occur rapidly after few days of high pyrexia, and they are prime factors leading to mortality and morbidity. If prompt diagnosis and appropriate supportive treatment have been given before and during the narrow window of shock/leakage and hemorrhage, the disease is self-remitting and rarely leads to fatality. Definitions of immune responses that lead to a protective immunity, versus those that cause DHF are still not known and are badly needed.

Utilizing database and specimens from cohorts of patients admitted to Khonkaen and Songkla hospitals (via the support of the Thailand Tropical Diseases Research Program – NSTDA/WHO/TRF), we have conducted for the last decade, a group of basic researches aiming at defining the type of immune responses and factors associated with the

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pathogenesis processes involved in dengue hemorrhagic fever. The roles of cell-mediated immune responses involved in different clinical phenotypes of dengue virus infection have been demonstrated; profound T-cell activation and death, and responses skewed toward cytokine production in the absence of degranulation, might contribute to systemic disturbances leading to DHF.

Monoclonal antibodies from human B cells isolated from DHF patients were shown to be specific against the dengue envelope, pr-M and the NS1 non-structural proteins. Anti-prM monoclonals poorly neutralize the viruses, but can ‚enhance‛ virus infection by combining with the presumed ‚immature-prM-containing‛ viruses and infect cells via the Fc-receptor. The finding has implications for future vaccine design.

More advance studies will be presented. Much progresses have been made when basic science has been carried out in concurrence with clinical studies. This emphasizes again the values of a good clinical environment from which basic science studies could be conducted with aims of achieving better understandings that could contribute to better diagnosis, treatment and prevention of the rapidly spreading dengue hemorrhagic fever.

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Deep Sequencing Technologies (DSTs): The Clinical and Research Applications

Chonlaphat Sukasem, B.Pharm, Ph.D.1, 2*

Unit of Virology and Molecular Microbiology1, Laboratory for Pharmacogenomics and Personalized Medicine2, Department of Pathology, Faculty of Medicine,

Ramathibodi Hospital, Mahidol University *Corresponding author Summary

During the last few years, Deep Sequencing Technologies (DSTs) have become widely available and cost effective. These new technologies herald the era of Next Generation (NG) DNA Sequencing and move us towards increasingly cost effective whole genome sequences and the benefits that will bring to a wide variety of applications. NG sequencing technologies are a greatly flexible set of platforms that can be scaled to suit different approaches. In terms of sequence coverage alone, the NG sequencing is a dramatic advance over capillary-based sequencing, but NG sequencing also presents important challenges in assembly and sequence accuracy due to short read lengths, method-specific sequencing errors, and the nonexistence of physical clones. These problems may be surmount by hybrid sequencing strategies using a combination of sequencing methodologies, by new assemblers, and by sequencing more deeply. NEXT-GENERATION SEQUENCING is driving growth within the basic and biomedical research communities as rapidly as the bases are being sequenced. All agree, however, that with the implementation of any new technology there is a balancing act of cost-quality-quantity. The potential applications are as numerous as the samples to be analyzed. Each next-generation platform

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is optimized for specific sequencing applications. As a consequence, these technologies can be applied to a wide variety of viral research questions. In this review, the author focuses on the use of DSTs for clinical applications for detection of viral quasispecies including HIV-1 drug resistance strains and pathogen discovery. As well as, discuss the clinical and research applications of de novo sequencing platforms and bioinformatics tools for data analysis. Sequencing knowledge: Past, Present and Future

In 1977 Fred Sanger and Alan R. Coulson published two methodological papers on the rapid determination of DNA sequence, which would go on to transform biology as a whole by providing a tool for deciphering complete genes and later entire genomes. The method dramatically improved earlier DNA sequencing techniques developed by Maxam and Gilbert published in the same year, and Sanger and Coulson's own 'plus and minus' method published 2 years earlier. The obvious advantages of reduced handling of toxic chemicals and radioisotopes rapidly made 'Sanger sequencing' the only DNA sequencing method used for the next 30 years.

Sequencing knowledge has made great advances over the last 30 years since the development of chain-terminating inhibitor-based technologies. For amplification and sequencing of individual templates using vector-based primers, conventional sequencing approaches need cloning of DNA fragments into bacterial vectors. This approach was adapted for cDNA libraries and, with the advent of capillary sequencing, became suitable for high-throughput sequencing of large samples of transcripts, termed Expressed Sequence Tags (ESTs). ESTs have become an invaluable resource for gene discovery, genome annotation,

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alternative splicing, SNP discovery, molecular markers for population analysis, and expression analysis in animal, plant, and microbial species.

New technologies of high-throughput DNA sequencing are sparking a revolution in the life sciences. Next generation sequencing (NGS), also called massively parallel (their capacity to process millions of sequences in parallel), is a highly parallelized approach for quickly and economically sequencing new genomes, re-sequencing large numbers of known genomes, or for rapidly investigating transcriptomes under different conditions. NGS technologies produce an unprecedented number of sequence fragments in the 20-300 base pairs [bp] range. Deep sequencing technologies: the next generation

In 2005, Next-generation sequencing methods were first introduced commercially. These new sequencing technologies, the 454 system using pyrosequencing technology, and the Solexa system detects fluorescence signals were introduced both based on sequencing by synthesis. In 2009, Applied Biosystems has introduced their SOLiD sequencer with read lengths anticipated to be 50 bp in the upcoming SOLiD3 release. In contrast to the conventional 96-capillary capacity of Sanger sequencing, these technologies execute millions of sequencing reactions in parallel, producing data at ultrahigh rates. However, the average read length of 454FLX and 454Titanium is 100–230 bp and 300–400 bp, respectively. For Illumina Solexa and SOLiD sequencer platforms, the average read length is 35-76 bp and 20–35 bp, respectively. Although the read lengths are much shorter with these new methods than with capillary sequencing both platforms generate sufficient data to completely re-sequencing genomes in a single run, which promised to replace or enhance traditional sequencing methods.

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Although individual read lengths are currently limited (<500 base pairs [bp] for most platforms), the depth of coverage per base pair and advanced sequence assembly software allow sequencing of 0.5–60 giga base pairs (Gbp). Thus, in a single run, depending on the platform, it is possible to sequence anywhere from one half to 53 times the size of the chicken genome, or a minimum of 3,000 mitochondrial genomes. These methods extend far beyond simply genome sequencing and have already greatly benefited the field of biology, leading to advances in evolution epidemiology, phylogenetic, comparative genomics, microbial diversity, DNA marker discovery, and studies of gene function and expression.

The three platforms offer a variety of experimental approaches for characterizing a transcriptome, including single-end and paired-end cDNA sequencing, tag profiling (3' end sequencing especially appropriate to estimating expression level), methylation assays, small RNA sequencing, sample tagging ("barcoding") to permit small subsample identification, and splice variant analyses. Several challenges face investigators hoping to use these methods, including the relatively large cost of most NG experiments and intense demands for data storage and analysis on the scale required for NG datasets, and rapidly evolving technologies.

Although even the instruments could easily generate a throughput equivalent to that of more than 50 capillary sequencers at about one-sixth of the cost, the reaction of the scientific community was surprisingly reserved. Instead of embracing the new technology and rapidly adapting to use its enormous potential, many scientists accustomed to using Sanger sequencing raised issues such as sequencing fidelity, read length, infrastructure cost or simply objected to the need to handle the large volume of data generated using the new

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technology. The initial solutions were strategies that mixed Sanger and

pyrosequencing data. As the cost and effort of the Sanger component in any project still is prohibitively expensive, many laboratories now rely solely on next-generation sequencing data or combine the advantages of relatively long reads from pyrosequencing with the low operating costs of Illumina's Solexa or Applied Biosystem's SOLiD platforms, thereby independently verifying each system's performance. With the availability of more non-Sanger sequencing methods, it now becomes possible to assess both the next-generation sequencing accuracy and the correctness of the vast majority of Sanger-based reference sequences in the public databases. Short read length and long read length

An increasing number of platforms are available for massively parallel sequencing. The platforms could be argued that provide short reads but with a higher throughput in terms of the number of molecules analyzed might be more efficient and cost effective than platforms that provide longer reads but with a lower throughput in terms of the number of molecules sequenced. In such applications, as long as the read length is sufficient for mapping back to the genome (e.g., 25–36 bp) or another reference set of sequences, the task would be adequately performed. On the other hand, for applications that are focused on the characterization of samples such as identification of novel pathogens or mutation detection, platforms with a longer read length might have advantages over platforms with shorter read lengths. With the rapid increases in read lengths and throughput of the various platforms, the gaps between these systems for plasma/serum nucleic acid sequencing might become narrower in the

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near future. It will probably still be a number of years before the diagnostic applications using massively parallel sequencing become commonplace, however.

Application of deep sequencing technologies for viral researches and clinical aspect

With highly mutation-prone viruses like HIV, hepatitis C virus, or influenza, our understanding of genome sequences has been based on the overall average genome (the average of a vast and diverse population). That average have been calling the genome of these viruses, may not even exist as such, and certainly the minor variants that have been missed by traditional methods are also critically important, because they can explode out within a few days to take over the entire population, given the right set of circumstances. For example, if among those minor variants there are a few drug-resistant strains, then as soon as you treat the host, those variants may be able to take over.

At this time, the recent development of deep sequencing technologies may facilitate a better understanding of the genetic composition and natural evolution of viral quasispecies in the presence of antiviral drugs. Currently, the recombinant phenotypic assay is most widely utilized; however, genotypic tests may represent alternative methods. Ultra-deep pyrosequencing generated data with some order of magnitude higher than any previously obtained with conventional approaches. Next generation sequencing allowed the analysis of previously inaccessible aspects of HIV-1 quasispecies, such as co-receptor usage of minority variants present, for assessing HIV tropisms (CCR5 or CXCR4).

The application of next generation sequencing to HIV research is

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extremely powerful because the virus rapidly mutates as a part of its normal biology. HIV-1 drug resistance is one area in particular that will benefit from the application of next generation sequencing. Low abundance drug resistant HIV variants at levels as low as 1% of the circulating viral quasispecies can be detected in antiretroviral (ARV)-naive individuals by sensitive and quantitative genotyping technologies. These low abundance drug resistant variants have been shown to potentially impact clinical responses in individuals initiating non-nucleoside reverse transcriptase based ARV therapy. However, the detailed study of the dynamics of viral variants present in a quasispecies population has long been hampered by the lack of sensitive sequencing methods. Indeed, studies of HIV suggest that these more-sensitive sequencing technologies detect additional minority variants for both treatment-naive and treatment-experienced patients which could impact the clinical outcome of antiretroviral therapy and may provide important information for treatment planning. The choice of anti-retroviral treatment based on the genetic make-up of the viruses infecting an individual is a real possibility through the deep sequencing of the viral genomes present. Information regarding potential drug resistant viruses can be used by the physician to choose the most effect treatment course. The next generation sequencing technologies offer the combination of speed, cost and accuracy demanded to meet the growing need for the genetic analysis of patient populations.

Advancements in molecular medicine due to next generation sequencing is not limited to the HIV drug resistance testing, but also deep sequencing technologies also enabled the study of minority variants present in the HCV quasispecies population present during antiviral drug pressure, giving new insights into the dynamics of resistance acquisition

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by HCV. Detection of HCV quasispecies is not the only area impacted by the application of new sequencing technologies. Throughout the pandemic 2009 influenza A virus (A/H1N1/2009) has emerged globally. De novo sequencing can comprehensively detect pathogens, and such in-depth investigation facilitates the identification of influenza A viral heterogeneity. To better characterize the A/ H1N1/2009 virus, unbiased comprehensive techniques will be indispensable for the primary investigations of emerging infectious diseases.

The massive throughput enabled by these platforms has allowed researchers to dig deeply into the metagenome of a viral population and identify all subtypes of virus present. The ability to sequence a viral genome thousands of times on a single sequencing run makes them an ideal tool for anti-viral research. As a result, these sequencing based technologies will have a major impact on viral researches, including the development of diagnostic and prognostic assays, the identification of altered proteins to which targeted therapies may be developed, the ability to predict onset and severity of disease, viral researchers are using next-generation sequences to identify unknown etiologic agents in human diseases, to study the viral and microbial species that occupy surfaces of the human body, and to inform the clinical management of chronic infectious diseases, and an improved capability to predict our range of responses to pathogenic agents. Pharmacogenomics and theranostic markers

There are two competing forces in drug discovery and development; (1) the desire to produce drugs with broad applicability and (2) the need for increased specificity and safety of new drugs. New technologies are required that increase specificity with no significantly

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rising drug development costs to help mediate these two forces. There are brilliant examples of compounds with good economic profiles that are effective for patients with specific genetic profiles such as Herceptin (Genentech), Iressa (AstraZeneca) and Gleevec (Novartis).

Incorporation of DNA sequencing into the drug discovery method will permit the identification of specific patient populations as well as identifying diagnostic and/or theranostic markers. DNA sequencing offers the most reliable and accurate method of grouping individuals into characteristic genetic profiles. Sequencing of disease-associated regions enables the differentiation of genetic profiles, regardless of the underlying genetic changes. Thus far, DNA sequencing has been of restricted use in clinical trials because of the unaffordable cost and amount of time associated with sequencing the hundreds of individuals enrolled in a single trial.

Nowadays, the economics are shifting in favour of sequencing because of the massive throughput of next generation sequencing. Parallelisation of samples processing can further facilitate the stratification of patient populations via the analysis of specific genomic regions. The pharmaceutical industry is on the cup of experiencing clinical trials stratified by the genetic profile of its subjects. Next Generation Sequencing is suspended to enable this paradigm shift through fast, inexpensive, and accurate sequencing. The ability to stratify clinical trials will reduce the cost of getting drugs to market and will make drugs more specific to their target populations. The application of these technologies in the future of pharmacogenomics, as well as follow-on patient care, should provide significant improvement to the process including patient stratification and identification of disease specific genetic markers. Can it be applied for research and clinical application now?

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With the publication of more than 100 research articles in less than two years, next-generation sequencing has demonstrated its enormous potential for anyone working in the life sciences, at a time when many believed the age of post-genomics had arrived. It also has brought the field of genomics back into the laboratories of single investigators or small academic consortia, as is evidenced by the fact that the majority of next-generation sequencing publications originate from sites other than the large genome centers. One therefore will wonder, when looking back from the not too distant future, why the application of next-generation sequencing technologies initially was not more cheerfully welcomed in the scientific community and, more importantly, by the public funding agencies. Hopefully this lesson will have been learned when the 3rd generation of sequencing instruments is introduced, as by then the success of the current initiatives should have broken the ice that 30 years of Sanger sequencing have cast over the sequencing landscape. Clinical setting of Next generation technologies: The list of improvements

Despite having already enabled a superfluity of studies by means of next-generation sequencing, scientists and engineers who are working on these tools and the companies that commercialize the applications still have to do list of improvements;

1. High on the list is cost reduction: a reduction of 1–2 orders of magnitude is needed to deliver on the promise of personal genomics, which targets a cost of $1,000 for the resequencing of a human genome.

2. Additionally, a reduced sequencing error rate would be highly welcome, not only for all present next-generation sequencing

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technologies, but also for Sanger sequencing, which clearly will continue to make valuable contributions in the immediate future. This might come in the form of custom-tailored DNA polymerases that provide a direct readout of DNA sequence in the form of emitted light, but even with these improvements we are unlikely to see a digital translation of DNA sequence into machine-readable code.

3. Currently, the equipment and reagents are still relatively expensive; a reduction of machines would be highly greeting.

4. Furthermore, the bioinformatic support that is needed to analyze the data is enormous and out of reach for most diagnostic laboratories at the present time. As cost comes down, the amount of data are likely to missile, creating an analytical bottleneck. Therefore much of the gain provided by future generations of sequencing instruments will be compensate by increased costs and efforts on the bioinformatics front. Therefore, expand the advance courses of bioinformatics, leading to gain the advance bioinformatician is highly recommended.

Conclusion

The importance of this new technology is exemplified by the fact that more than 120 studies have been published since the introduction of the first next generation sequencing technology. Publication of novel information is a significant validation for the technology and many of the applications have direct relevance to drug discovery and development. Importantly, the next generation sequencing has facilitated new research approaches including the whole genome analysis of disease causing

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organisms, the extremely deep analysis of drug resistance mutations in complex genetic settings such as HIV, the genomic characterization of a community of micro-organisms (metagenomic) from diseased versus healthy individuals to elucidate a causative agent, the broad genetic analysis of drug targets across a population and the better understanding of chromatin organization and epigenetic regulation of gene expression. The new sequencing platforms’ capacity to address many therapeutic areas will materially affect the price of drug discovery across organizations. The next generation sequencing technologies allows for applications across many different experimental areas. Nonetheless, with additional technical advances and cost reduction in the coming years, it is highly likely that massively parallel sequencing approaches will eventually become a routine tool in laboratory medicine.

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