Advances in Virus Research: 51

Virus taxonomy in the age of metagenomics
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The method builds upon recent studies in which we reported the construction and characterization of PRV [17] and demonstrated the ability of lentivirus mediated Cre expression to produce conditional reporter expression from a Brainbow cassette [19] carried by PRV in targeted populations of neurons [18]. Here we describe the construction and use of PRV, which serves both as a transneuronal tracer and a vector for circuit related expression of Cre.

To our knowledge this is the first demonstration of the ability to deliver biologically active Cre in a circuit specific fashion across multiple synapses. The fact that Cre is expressed throughout the polysynaptic circuit infected by PRV is validated both by the pattern and kinetics of conditional reporter expression observed within the CNS following separate injections of PRV and PRV into the kidneys. Importantly, the present data confirm the identity and organization of neurons within the preautonomic network previously shown to collateralize to regulate both kidneys [20].

Considered with evidence that recombination of the Brainbow cassette only occurs in the presence of Cre, this is an important confirmation that PRV is producing biologically active Cre in the neurons that it infects. Importantly, the insights derived from the use of PRV and PRV in dual infection experiments are not limited to the ability to identify neurons that collateralize to influence separate targets.

These neurons can only have been infected subsequent to Cre mediated recombination and transneuronal passage of the PRV genome. Using in vitro analysis Kobiler and colleagues demonstrated that Cre-mediated recombination occurs prior to replication of PRV and that a remarkably small number of viral genomes — as few as seven — are expressed, replicated and assembled into virions [17]. This interesting bottleneck may limit the population of virions that can spread transneuronally and express their genomes.

In any case, even if PRV and PRV co-infect a single neuron, the data of Kobiler and colleagues indicates that the probability of second- and third-order neurons being infected by both recombinants drops after each transneuronal passage. Therefore, neurons displaying only cytoplasmic reporters of the recombined PRV genome, and no reporters of PRV infection punctate VPRFP , likely represent neurons that were infected from the early transneuronal passage of progeny virus containing the recombined PRV genome from a dual infected neuron.

Similarly, early transneuronal passage of PRV, and not PRV recombinants, from dual infected neurons would produce neurons only expressing the PRV genome that are indistinguishable from neurons connected only to the PRV infected kidney. Thus, data derived from this approach must be interpreted conservatively and conclusions on the synaptology of the circuit based only upon positive unequivocal results.

In this regard, the singular expression of PRV reporters of the recombined Brainbow cassette provides an unambiguous identification of neurons presynaptic to dual infected neurons.

Feedback Circuits in Hepatitis B Virus Infection

As noted above, the in vitro data of Kobiler and colleagues demonstrated that Cre-mediated recombination of the PRV genome occurs prior to replication of the virus. However, there is a chance that several incoming PRV genomes will initiate replication before recombination can occur, even in the presence of PRV This can result in neurons that were infected with both viruses expressing the default dTomato reporter along with the reporters liberated by Cre mediated recombination.

Under these circumstances it is possible that a single dual infected neuron can replicate up to four different viral genomes PRV, PRVred, PRVyellow, and PRVblue and transneuronal infection of synaptically connected neurons would sample any combination of these replicated genomes.

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Indeed, we often observed neurons in vivo that expressed dTomato a reporter of the uncombined PRV genome along with the mCerulean and EYFP reporters of recombination. The functional implications of being able to identify neurons presynaptic to collateralized neurons are apparent in our data. The neurons identified in their investigation are among the dual infected neurons observed in our investigation and include areas that have been identified as important mediators of neural responses stress.

Nevertheless, the LC is prominent among the cell groups activated by stressful stimuli and it has been postulated to play a prominent role in orchestrating behavioral and physiological responses to stressors [33] , [34] , [35]. Importantly, available evidence indicates that the LC does not exert its influence upon sympathetic outflow through direct reticulospinal projections to SPNs in the IML [36].

Rather, LC neurons project to components of the preautonomic network that, in turn, project directly to SPNs e. Our data suggest that the LC contains a large population of neurons presynaptic to dual labeled neurons, an observation consistent with a prominent role for the LC in the global activation of sympathetic outflow that is a cardinal feature of the fight-or-flight response.

Our data are also consistent with a similar functional role for the hypothalamic PVN, which also contained prominent populations of neurons presynaptic to collateralized neurons. Definitive support for these hypotheses requires quantitative analysis of a larger sample size, but the possibility illustrates the potential power of the combined use of PRV and PRV in dual infection analysis of neural circuitry.

The ability to express Cre in a circuit related manner through PRV infection and transneuronal passage also has other experimental applications. For example, PRV can be used to mediate recombination of floxed genes in transgenic mice in a circuit-defined manner. Given the expanding list of floxed genes that are widely available e.

Additionally, the virus can be used to produce circuit related conditional reporter expression in the nervous system of the Brainbow mouse [19]. In conclusion, we have described a new viral tracing method based on the polysynaptic tracing properties of PRV, the ability to express biologically active Cre from the PRV genome, and the conditional reporter capabilities of the Brainbow cassette. The method enables identification of neurons that collateralize within a complex network to exert regulatory control over distant separate targets.

It provides a means of expressing Cre in a circuit specific fashion from a replication competent PRV recombinant PRV and relies upon Cre-dependent combinatorial expression of fluorescent reporters from a Brainbow cassette carried by second PRV recombinant PRV The unique reporter phenotypes produced in dual infection studies employing these recombinants provides unique insights into the synaptic organization and function of polysynaptic networks and increases the diversity of viral transneuronal tracing tools available for circuit analysis.

We thank Jeff Lichtman for the brainbow plasmids and acknowledge members of the Enquist and Card laboratories for advice and technical assistance. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Replication and transneuronal transport of pseudorabies virus PRV are widely used to define the organization of neural circuits in rodent brain.

Introduction Neurotropic viruses represent popular and powerful tools for defining the identity and organization of synaptically connected neurons [1] , [2] , [3] , [4] , [5] , [6] , [7] , [8] , [9].

Mining viral genomes to improve clinical diagnostics

Animals Adult male rats Harlan Sprague-Dawley weighing to grams at the time of viral injection were used for in vivo experiments conducted in a Biosafety Level 2 certified laboratory. Download: PPT. Experimental design The design of the experiment is illustrated in Figure 2A. Immunoperoxidase localizations The invasive profiles of the recombinants were first determined by immunoperoxidase localization of infected neurons. Fluorescence microscopy Sections of brain and spinal cord adjacent to those used for the immunoperoxidase analysis were analyzed using fluorescence microscopy.

Data analysis We first characterized the extent of viral invasion of renal presympathetic circuits using immunoperoxidase localization of infected neurons in brain and spinal cord. Results Our experimental design takes advantage of a well characterized dual infection paradigm that results in predictable retrograde transneuronal passage of PRV recombinants from the kidneys [20].

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Fluorescence profiles of infected neurons Neurons infected by PRV express a red punctate signal that produces a dense labeling of cell nuclei early in viral replication Figures 2B followed by the appearance of red puncta in the cytoplasm later in infection Figures 2C. Reporter gene expression in neurons replicating only one recombinant Reporter gene expression in sympathetic preganglionic neurons SPNs of thoracic spinal cord illustrated the distinctive reporters of neuronal infection with PRV or PRV Reporter gene expression in dual infected neurons We analyzed regions of the spinal cord, medulla and diencephalon previously shown to contain neurons infected through collateralized axonal projections to neural circuits innervating each kidney [20].

Transneuronal infection from dual infected neurons The expression of conditional reporters of the Brainbow cassette throughout the preautonomic network, while confirming the presence of collateralized neurons, also revealed new insights into the synaptic organization of preautonomic synaptology. Discussion The findings reported in this manuscript document a new viral transneuronal tracing approach that can be used to identify connections to neurons within a complex network whose axons collateralize to influence separate targets.

Acknowledgments We thank Jeff Lichtman for the brainbow plasmids and acknowledge members of the Enquist and Card laboratories for advice and technical assistance. References 1. Advances in Virus Research — View Article Google Scholar 2. Mettenleiter TC Molecular properties of alphaherpesviruses used in transneuronal pathway tracing.

San Diego: Academic Press. Loewy AD Viruses as transneuronal tracers for defining neural circuits. Neuroscience and Biobehavioral Reviews — View Article Google Scholar 4. Progress in Neurobiology — View Article Google Scholar 5. Callaway EM Transneuronal circuit tractin with neurotropic viruses. Current Opinion in Neurobiology — View Article Google Scholar 6.

Journal of Neuroscience Methods 63— View Article Google Scholar 7. Card JP Pseudorabies virus neuroinvasiveness: A window into the functional organization of the brain. Advances in Virus Research 39— View Article Google Scholar 8. Nature Methods 6: — View Article Google Scholar 9.

Virus Research — View Article Google Scholar Sauer B Functional expression of the Cre-Lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Science — Cell View Article Google Scholar Campbell RE, Herbison AE Definition of brainstem afferents to gonadotropin-releasing hormone neurons in the mouse using conditional viral tract tracing. Endocrinology — Campbell RE, Herbison AE Defining the gonadotrophin-releasing hormone neuronal network: Transgenic approaches to understanding neurocircuitry. Journal of Neuroendocrinology — Journal of Comparative Neurology — Nature Communications 1: Nature 56— Cano G, Card JP, Sved AF Dual viral transneuronal tracing of central autonomic circuits involved in the innervation of the two kidneys in the rat.

Smith GA, Enquist LW A self-recombining bacterial artifical chromosome and its application for analysis of herpesvirus pathogenesis. PNAS — Journal of Virology — Smith GA, Enquist LW Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alpha herpesvirus.

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To help conceptualize the sheer number of viruses in existence, their current biomass has been estimated to equal that of 75 million blue whales approximately million tonnes and, if placed end to end, the collective length of their virions would span 65 galaxies 6. Grapevine vitivirus A eradication in Vitis vinifera explants by antiviral drugs and thermotherapy. The pentameric motor processively translocates DNA until the head shell is full with one viral genome. Kuo, G. Plants, viruses and the environment: ecology and mutualism.

Peptides 7: — Journal of Neuroscience — Amsterdam: Elsevier. Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization. Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs.

Nine isolates Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from to against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses.

Up to fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity R was calculated, 49 of 66 comparisons Moreover, one isolate had R values suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.

Key words: bovine viral diarrhea virus, BVDV, monoclonal antibodies, cross-neutralization, antigenic diversity. Bovine viral diarrhea virus BVDV is an important cattle pathogen which causes significant losses to the livestock industry around the world 1,2. Pestiviruses are small enveloped, positive-sense RNA viruses 4,5 which naturally infect swine and domestic and wild ruminants 6.

Pestiviruses display considerable genetic and antigenic diversity within the genus, although all members cross-react serologically to some extent 7. BVDV infections in cattle are associated with a variety of clinical manifestations including inapparent infections, gastroenteric, respiratory, and hemorrhagic syndromes and the deadly mucosal disease MD Infection of pregnant cows by the noncytopathic BVDV biotype may result in a variety of outcomes: early or late embryonic death, abortion or mummification, malformations, stillbirth and birth of weak and non-thriving calves Infection of fetuses between 40 and days of gestation often leads to fetal immunotolerance, resulting in the birth of immunotolerant persistently infected PI calves 9, Most persistently infected animals develop and die of mucosal disease within the first 6 to 24 months of life 2,9.

Cytopathic BVD viruses originate from their noncytopathic counterpart through mutations, recombinations or rearrangements in the viral RNA genome 11, Serological studies have shown that although BVDV field isolates are antigenically related to each other, antigenic differences can be readily detected among viruses 7, These antigenic differences have been demonstrated by the use of monoclonal antibodies mAbs and by in vitro and in vivo cross-neutralization studies 7, However, in spite of the antigenic variability, no clear definition of serotypes has been possible to date 7, The marked antigenic variability of BVD viruses allows differentiation between strains, but also represents a potential problem for diagnosis and immunization strategies 7, The presence of BVDV infection in the country was first indicated by clinical reports and serological studies conducted in the 60's and 70's Thereafter, serologic and virologic data have confirmed the widespread distribution of BVDV infection among Brazilian cattle 20, The present article reports the antigenic characterization of 19 of these isolates.

Analysis of reactivity with a panel of mAbs and cross-neutralization studies revealed a marked antigenic diversity among these viruses. This antigenic diversity may have important implications for epidemiologic studies and for the diagnosis and control of BVDV infection in Brazil. Material and Methods. Eleven viruses were isolated from the blood of bovine fetuses collected at slaughterhouses in the central region of Rio Grande do Sul State, southern Brazil. The other 6 viruses were isolated in other laboratories and sent for characterization Botton SA and Flores EF, unpublished data.

Rockville, MD and submitted to three blind passages at h intervals. At the end of the third passage, the inoculated cells were submitted to an indirect immunofluorescence assay IFA for viral antigens. The slides were counterstained with Evans blue, mounted with PBS:glycerol and observed in an epifluorescence microscope. The mAbs were provided by Dr. The ability of each individual mAb to recognize and bind to viral antigens was assayed by IFA.

Cells infected with each isolate were incubated with individual mAbs as the primary antibody, followed by incubation with the secondary antibody as described above. Either ascites fluid in phosphate buffered saline; PBS or supernatants of hybridoma cultures were used. Mock-infected cells, cells infected with the prototype Singer strain and stained with each individual mAb, and cells infected with each isolate and stained with a pool of mAbs were used as controls. Eleven BVDV-seronegative, 6- to 8-month-old lambs were inoculated intranasally and intramuscularly with approximately 10 7 TCID 50 tissue culture median infectious dose of each virus.

The animals were housed individually until the second serum collection. Blood was collected at 15 and 30 days post-inoculation pi to obtain serum which was heat inactivated at 56 o C for 30 min prior to virus neutralization VN assays. Individual serum samples collected at 15 and 30 days pi were initially titrated against their homologous viruses in a standard VN assay The serum samples obtained at 30 days pi which had the highest VN titer were titrated against each heterologous virus and the neutralization end points were determined for each virus-serum combination.

VN assays were performed in polystyrene well plates, using doubling two-fold dilutions of each serum starting at Readings were performed after 96 h of incubation. Whenever a certain serum was titrated with different viruses, the VN tests were performed at the same time on the same plate, using the same preparation of MDBK cells. The cross-neutralization tests yielded values, corresponding to the VN titer of each virus-serum combination.

These values are reported as the reciprocal of the highest dilution of serum capable of preventing viral replication Table 2. The data obtained in the cross-neutralization assays were combined in order to calculate the antigenic similarity among the isolates. The neutralization end points for each virus-serum combination were combined as described by Howard et al. The coefficient of antigenic similarity R was calculated according to Archetti and Horsfall 23 using the following formula:.

BVD viruses were isolated and identified in 0. One additional virus was isolated from a clinical case of gastroenteric disease and another from the serum of an apparently healthy calf from a dairy herd. All these isolates were of the noncytopathic biotype. The origin and preliminary characterization of these and other 6 viruses isolated in other laboratories will be described elsewhere Botton SA and Flores EF, unpublished data.

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The protein specificity of the mAbs used in this study and a summary of their reactivity with Brazilian BVDV isolates are presented in Table 1. Except for a mAb that recognized 18 Four mAbs of undetermined protein specificity displayed a variable spectrum of reactivity recognizing from Virus-specific antisera produced in lambs against three BVDV standard strains and 8 native isolates had moderate to high levels of neutralizing antibodies. Bioinformatic platforms are key components of the sequencing process.

They allow interpretation of the sequencing output through computational analysis Naccache et al. Pyrosequencing is currently the variant of choice within NGS systems. Based on the detection of pyrophosphate PPi after incorporation of a nucleotide in a DNA polymerization process, it uses luciferase to catalyse light-generating processes, and the collected light is then recorded. Although Illumina is considered the most frequently used pyrosequencing platform, FLX; a subsidiary company of Roche, was the first high-throughput analyser in the market and was used to determine human papillomavirus HPV types Barzon et al.

Other variants detect hydrogen ions that are released throughout nucleotide incorporation reaction Rothberg et al. Generating high volumes of sequence data has allowed the compilation of viral nucleotide databases and acquisition of de novo sequences to understand the genetic variability of viruses Szpara, Parsons and Enquist, HIV is by far the most sequenced because of the global priority of AIDS as a serious endemic, and because of the high mutation rate of the virus.

So far, only gene sequencing methods have been successful in genotyping tricky HBV, unlike conventional PCR or serological assays. Therefore, sequencing has allowed better clinical management of HBV infection and related complications Margeridon-Thermet et al. In data analysis, recent technical approaches have included adjustment of the software reading platforms for simultaneous detection of genotypes and mutants of clinical importance Germer et al.

Nucleotide sequencing of HCV sub-genomic regions is now the method of choice for genotyping. The principal requirements for NGS are initially access to a sequencer, and considerable skills in bioinformatics and expertise in data analysis, plus adequate handling systems for storage of generated data.

Adding to that, despite the outstanding results delivered by prototypes in trials, many are still at the research level, and not yet approved for use in routine clinical practice Jiangqin et al. NGS is undeniably a key technology in specialized clinical laboratories, but its implementation is still a challenge in many countries, where not only their resource-limited settings cannot afford a sequence analyser, sample and library preparation, but the vast majority of the population cannot afford the cost of the test.

Mass spectrometry MS is nowadays a benchmark of laboratory qualitative and quantitative investigation, particularly in bacteriology Sauer and Kliem, The principle of MS relies on converting the sample into charged particles ions by ionization process. The result obtained is compared to a reference database library , existing within the system and delivered as an interpretive spectrum.

In clinical laboratories, matrix-assisted laser desorption ionization MALDI and electrospray ES are the most used ionization methods because they allow processing of considerable amounts of analyte Emonet et al. These approaches have extensively been evaluated experimentally and provided excellent results, either used alone or combined with other molecular methods, such as PCR, in order to enhance sensitivity.

This blend of two powerful machineries PCR-MS can detect drug resistance to antiviral therapy as well as the presence of multiple viruses within the same sample and diagnose for co-infections, when assays are multiplexed. The same method was applied for genotyping and successfully detected the eight HBV genotypes accurately Ganova-Raeva et al. Mass spectrometric-based methods are versatile, sensitive, rapid and cost-effective, and do not require interpretation software for data analysis. The automated machinery necessitates easy sample preparation and fewer operators. Tests can also be performed efficiently on archived specimen.

The main limitation of MS is the high cost, particularly in high pandemic areas, which are usually the poorest; not all laboratories can afford a mass analyser for their activities. The second major drawback is within the reference library. The identification is limited by known data from well-identified organisms only; therefore, rare mutations cannot be detected if they do not exist within the reading platform, but there is hope that MS database libraries will rapidly expand.

Small sample volumes are needed, so the assays can still be performed in particular cases CSF, new-borns, etc. Some assays such as MS remain efficient after several freezing—thawing of the samples archived samples. Rapid TAT: minimum time required from sample collection to results reporting is 30 min, and microarray tools can deliver results within seconds. The methods described above have shown outstanding performance in saving thousands of lives in developed countries. However, accurate diagnosis is still a challenge in resource-limited settings because of the difficulty in acquiring these equipment and technical expertise.

Ultrasensitive p24 Patton et al.

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Unfortunately, their lack of sensitivity has induced failure of treatment and emergence of resistant strains Vekemans, John and Colebunders, Hepatitis is another expensive burden to manage in poor countries, where accurate assessment of the epidemiological profile is hard to establish. These low-cost devices do not allow determination of the course of the disease to inform whether to initiate an antiviral treatment or not.

Providing high-level epidemiological monitoring of viral diseases is undeniably a global public health ambition, and despite rapid progress in the development of diagnostic methods in recent years, improvements are needed for better cost, size Loman et al. After the Ebola Haemorrhagic fever in , the US governmental Department of Energy has developed an innovative rapid and portable test to detect specifically Ebola virus within seconds.

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The test aims to target other RNA and exotic viruses such as Dengue and West Nile for effective management of viral outbreaks. Microfluidic technology is regarded with optimism for the management of infectious diseases by allowing timely therapy. It provides a key potential solution for remote areas and near-patients facilities by avoiding turnaround trips of the patients between the clinic and the laboratory. Their use is also beneficial where time is crucial, or when physical spaces do not allow setting up of conventional methods.

Lab-on-a-chip LOC is a very small device that integrates laboratory processes within a few square centimetres. It uses very small volume of samples to perform immediate reactions within the chip or in a portable device. The reactions vary from nucleic acid amplification and detection, to cell count and immunoassays; therefore, microfluidic diagnostics compete with large instruments in performing laboratory tests at a lower cost, to benefit low-income settings and remote areas. The bench top analyser GeneXpert made by Cepheid has an integrated sample preparation and PCR system for molecular diagnosis of influenza and other bacterial infections in a light portable format.

Improvement of QC programs, QA and standardization of assays, kits and reagents are important to fulfil requirements for accuracy. The recently developed viral diagnostic methods are reshaping the field of clinical microbiology, and could contribute to reducing the prevalence of serious infectious diseases. However, the technical capabilities alone are insufficient if not supported by health promotion strategies to increase awareness about the importance of early detection and regular screening of persons at high risk.

Finally, good quality diagnosis has a cost that only developed countries can afford in routine practice so far, and this is delaying the implementation of new methods in the developing world and the endemic areas. However, there is hope that efforts will continue towards developing new good quality tests affordable in low-income countries, which would substantially strengthen disease control strategies for their populations. She works as a biomedical scientist at Institut Pasteur, within the Immunoassay division, and has particular interest in laboratory diagnosis issues in low-income settings and how they can be solved at lower cost.

She also has interest in structural biology and microbiology. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search. Article Navigation. Close mobile search navigation Article Navigation. Volume 9. Article Contents. General principles of good laboratory testing.

Traditional laboratory methods for the diagnosis of viral infections. Recent methods in the diagnosis of viral infections. Immunoassay-based tests. Amplification-based assays. Next-generation sequencing. Mass spectrometry. Advantages of the recent methods. The latest technologies and resource-limited settings.

Automation, microfluidics and future prospects. Author biography. Recent advances in diagnostic testing for viral infections Selma Souf. Oxford Academic. Google Scholar. Cite Citation. Permissions Icon Permissions. Abstract Viral infectious diseases represent an important portion of global public health concerns with thousands of deaths annually.

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