Introduction· Viral Nanotechnology provide general idea about fast developing in the field ofimmunology, virology, microbiology, chemistry, physics, and mathematicalscience. Its role is by leadingresearchers and practitioners, making it both inclusive and essential resources for study and research.· by increasing demand of the applications the viralnanotechnology is quickly expanding · Viral nanotechnology characterized Similarly as An note worthyscience that concerns itself with how toutilize the sub-atomic modules thoseunique separate science of molecular engineering only constructs. chances torevolutionize practices in energy, biomedicine, health care, photonics,catalysis, electronics, and public health are diverse by present potentialapplications of viral technology · the demand for newmethods and techniques in theprevention, diagnosis, and treatment of disease is considered as reasons forfast growth of Viral Nanotechnology.
There is also great application to be usedas methods for diagnostics, including the development of diagnostic substancesand novel imaging technologies for detecting disease and infectious agents (1) II. What is Virus?Viruses are small infectious agent that can exist everywhere ,their diameters in nanometers level. it’s found in air, soil animals and human bodies. but most of them are harmless. Human immunesystem protect the body against virus by generating antibodieswhich will recognize the virus and destroy it.The viruses can break thebalance and cause problems, diseases when the human immune system is weak dueto multiple reasons, so called opportunist pathogen.Viruses depend on the hostfor their survival and reproducing.
Viruses have many important functions forhumans, plants, animals, and the environment. such as some of them protect the host against otherinfections , by transferring genes among different species. In biomedicalresearch, scientists use viruses to insert new genes into cellsStructure of a virusVirus particle composed of threemain parts:· Nucleic acid ( DNA or RNA ) core of the virus DNA or RNA holds all of the information forthevirus and that makes it unique andhelp to multiply.· Protein Coat (capsid) –coveringover the nucleic acid which protect it.· Lipid membrane (envelope) – which covers thecapsid. naked viruses haven’t envelop Life cycle of a basic viruslytic cycle include :1. Adsorption process in which virus particleattaches to a host cell.
2. Entering DNA or RNA by inject the host cell.3. replication process by cellular enzymes which start making new virus particles 4. Relaease of the virus by newly formed viruses that kill the cell then andsearch for a new host cell. III.
Different Types of Virus.There are four main morphological virus types:Helical· It composes of a single capsomere stacked around a central axisto form a helical structure· rod-shaped or filamentous virions· short and highly rigid· long and very flexible.· The genetic materials, (DNA or RNA ), single-stranded RNA, butssDNA in some types , connected to the protein helix by interactions betweenthe negatively charged nucleic acid and positive charges on the protein.· Overall, the length of a helical capsid is related to the lengthof the nucleic acid contained within it· examples as tobacco mosaic virus IcosahedralMost infections viruses are icosahedral or close spherical withchiral icosahedral symmetry. A normal icosahedron is the ideal method forforming a close shell from sub-units. each triangular face has 3 identical capsomeresthat give 60 forit. Numerous viruses as rotavirus, have more than 60 capsomers and sphericalshape.Capsomeresare surrounded by hexonsthey might be made out of various proteins.
EnvelopeA few types of viruses covered themselves one of the celllayers, either the external film encompassing a contaminated host cell orinternal layers, for example, endoplasmic reticulum or nuclear layer get external lipid bilayer known as a viral envelope. This film is decorated by proteins coded for viral genome and host genome; The flu and HIV virusesutilize this techniqueComplexThese infections have a capsid that is neither helical noricosahedral, and that may have additional structures, as protein tails or acomplex external layer. for example, Enterobacteria phage T4, have bogglingcomposition comprising of an icosahedral head to a helical tail, which may have a hexagonal base plate with projectingprotein tail Virus has two types, DNA virus and RNA virus. The genome of DNAvirus is consisted of DNA, and similarly RNA is the genetic material of a RNAvirus. IV.
Effect of Virus on human Health Living organisms as animals,plants, fungi, and bacteria are all subject to be infected by viral infection. HoweverViruses select specific type of cells which want to invade. What does the role of host in order to fight off a virus invasion ? “immune response.” whichis created by Immune system produceantibodies to protect the host against any foreign substances.
Antibodies arespecific to each type of intruder, and new set of antibodies will have to be formedfor each new disease of infection. This process take several days. In the meantime,”interferons.
“which is produced by the cell infected by virus immediately to protect adjacentcells production of antibodies.the benefit of interferon in viral treatment isstill under research and the mechanismstill unknown.in case if viral infection ,antiviral infection can be prescribed.
List ofdiseases caused by viruses V. Diagnostic Methods of viruses 3 categories:(1) Direct detection(2) Virus isolation(3) Serology.1. Direct Examination of SpecimenClinical specimen examined directly for the presence of virus particles,virus antigen , and viral nucleic acids.
· Electron Microscopy morphology /immune electron microscopy· Light microscopy histologicalappearance – e.g. inclusion bodies· Antigen detectionimmunofluorescence, ELISA etc.· Molecular techniques for thedirect detection of viral genomes 2. Indirect Examination = VirusisolationØ Cell Culture· Cytopathic effect· Haemadsorption· Confirmation by neutralization,interference· Immunofluorescence etc.
Ø Eggs pocks – haemagglutination, inclusion bodiesØ Animals disease or death confirmation by neutralization 3. SerologyTitres increaseof antibody between acute and convalescent stagesof infection , detection of IgM inprimary infection.Classical Techniques · COMPLEMENT FIXATION TESTS (CFT)· HAEMAGGLUTINATION INHIBITIONTESTS· IMMUNOFLUORESCENCE TECHNIQUES(IF)· NEUTRALIZATION TESTS· SINGLE RADIAL HAEMOLYSISNewer Techniques· RADIOIMMUNOASSAY (RIA)· ENZYME LINKED IMMUNOSORBENT ASSAY(ELISA)· PARTICLE AGGLUTINATION· WESTERN BLOT (WB)1.Virus IsolationForvirus isolation cultured cells, eggs and laboratory animals are used. Cellcultures more widely used for virus isolation in many laboratories.For cellculturespreparation,tissue fragments are first dissociated, by using trypsin or collagenase. The cell suspension isthen placed in a flat-bottomed glass or plastic container (petri dish, a flask,a bottle, test tube) together with a suitable liquid medium.
e.g. an animalserum. After a variable slack, the cells will attach and spread on the bottomof the container and then isolation is started.
Primary and Secondary CulturesPrimarycultures are performed by replacing the fluid two or three times per week .thecells are separated from the vessel wall by either trypsin or EDTA since thecultures become too crowded, and the remaining portions are used to initiatesecondary cultures.Inboth primary and secondary cultures, the cells keep some of theircharacteristics from which tissue they are derived.Cellcultures are separated into 3 types:-1.
Primarycells -which are derived from animal or human tissues and can be sub-cultured only onceor twice e.g. primary monkey.2. Semi-continuousdiploid cells – which are prepared from human fetal tissue and can be sub-cultured20 to 50 times e.g. human diploid fibroblasts .3.
Continuouscells -originate fromhuman cancer cells oranimal tissue e.g. Cellcultures vary greatly in their susceptibility to different viruses. precautionsin cell culture :· Transportation of Specimens to thelaboratory should be as soon as possible once it’s taken.· Swabs should be placed in a bottle containingvirus transport medium.
· Bodily fluids and tissues should be kept in asterile container.¢ Identificationof a specific virus grown in infected cell cultures can be performed byneutralization of infectivity, hemadsorption inhibition, and mmunofluorescence. Effects of productive viralreplication in cell culture:· Cytopathiceffect(s) (CPE) as in mumps and measlesviruses· Syncitia(cell fusion)· HemadsorptionAdvantages of cell culture for virus diagnosis :· Relativeease· broadspectrum · Sensitivity.
Limitations:· Longperiod (up to 4 weeks) required for result.· Susceptibleto bacterial contamination.· Susceptibleto toxic substances which may be present in the specimen.· Manyviruses will not grow in cell culture e.g.
Hepatitis B, Diarrheal viruses,parvovirus, papillomavirus. · Difficultyin obtaining cell cultures.2. Electron MicroscopyIt’s used for detection andidentification of virus’s morphology by EM.Advantages:· Visualizationof virus· Directidentification of virus · Rapiddiagnostic toolDisadvantages:· Difficultto analyze multiple specimens· Availableof minimum number of virus (around 106 virus particles per mlfor detection)· Detectionis difficult for some viruses as SRSV· Expensive· Highlyskilled personnel are required.Types of EM methods;-1. DirectEM:negativestaining is used, small special equipment is required , Samples should be concentrated prior negativestaining by different methods as differential centrifugation,ammoniumsulphateprecipitation2. Immunoelectronmicroscopy (IEM).
High sensitivity and specificity It’s used in the following situations:· Smallnumber of virus particle are available.· Viruswhich have different morphological shapes as herpes viruses and picornaviruses· Inan outbreak conditions3.Haemagglutination Inhibition Testvirusesdirectlyagglutinateerythrocytes by bindingto specificreceptorsites on thesurfaceof theerythrocyteandthischaracteristiccan be used in detection,identificationandquantitationof thevirusSome virus will have ability tobindto erythrocytes (red blood cells), causing the formation of a lattice. This characteristicis called hemagglutination, Antibodies against the viral protein prevent virus to agglutinatethe erythrocytes , it’s known haemagglutination-inhibition test (HAI)It’s widely used for the diagnosisof rubella and influenza virus infections.Advantages:· Rapidin detection and identification of virus(few hours)· Cheap · Easily preparedDisadvantages:· Non- specific· low sensitivity 4. ELISA (enzyme-linkedimmunosorbentassay ) enzyme-linkedimmunosorbent assay (ELISA) was developed in 1970 ,also known as solid-phase enzyme immunoassay.
It is biochemicaltechnique mainly used to detect the presence of specific antibody or antigen inthe sample. It has been used as marker for disease diagnosis.Varioustechniques can be used in ELISA, the most important one are:1. Competitive assay2. Sandwichassay3. IndirectassayAdvantages:VerysensitiveQuantitative(a) Competitivemethod§ It’sAg-Ab complex.§ competitivereaction between antigen bound with primary antibody in plate and antigen existin the sample.§ highlysensitive.
§ Intensity of color is inversely proportional tothe concentration of antigen present in the sample.(b) Sandwichmethod§ Insolubilized antigen binds to theanalyte (the antibody) in the samplespecifically,§ addition of labelled enzyme linkedsecond antibody that binds to primary antibody.§ Unbound antibody-enzyme conjugates are washedoff.
(c) Indirect assay§ It’sused for detection of antibody present in the sample.§ Primaryantibodyexist in the specimen bind specifically to the antigen§ toremove unbound antibodies wash the solution.§ enzymewill conjugate secondary antibodies.
§ colorchange after addition enzyme substrate.§ Intensityof color direct proportional to concentration of primary antibodies.§ Cross-reactivitymight occur§ Anextra incubation step is requiredAssayCharacteristics :1.
Higher sensitivity; – either by selection of antibodies with a extremely highaffinity, or by reduction of the height and variability of the backgroundreaction, which makes very low concentrations of analyte more readilydetectable.2. Higher specificity;- by avoiding the presence of any antibodyin the assay system with specific reactivity against non-analyte epitopes, andby selecting combinations of monoclonal antibodies which may further increasespecificity.3. Higher practicality; – e.g.
by introducing simultaneous incubation of label,solid phase and sample without risk of “prozone effect”.5. Single Radial Haemolysis Single radial haemolysis (SRH) is usually used for the detection of rubella-specific IgG. Multiple serum can be analyzed simultaneously to confirm the immunity by SRH sensitive, specific, and reliable. 6. Immunofluorescense Immunofluorescence(IF) is commonly used for rapid analysisof virus infection.basicprinciple by using a fluorescein- labelled antibody to stain specific virusantigens present in the samples, so that the stained cells fluoresces under UVillumination.
Direct IF, the specimen is probed directly with a specific labelledantibody against specific virus antigen.Indirect IF, the specimen is first probed with anon-labelled specific antibody, followed by a labelled antibody against thefirst antibody.it has extra amplification step.§ Detectionof virus antigen can be done by direct IF or indirect IF§ Detectionof virus antibody can be determined always by indirect IF7. NeutralizationAntibodywhich protect the body from any infection can bind with virus to loss theinfectivity.
un-neutralizedvirus available may be detected by some techniques such as CPE, haemadsorption / haemagglutination.Thereare two types of neutralization:· Reversible neutralization DilutionAg-Ab complex within 30 min of the formation is defined as reversibleneutralization.· Stable neutralization This processis stable for long time ( several hours ) . It’s can’t be reversed by dilution.antibodies or virion don’t change.8.
Molecular Techniquesvarious molecular techniques havebeen developed in the last 10 years for detection microorganisms. these methodshave high sensitivity and specificity than conventional techniques.several techniquesused in molecular detection of virus such as : (a) PolymeraseChain Reaction(PCR)PCR based on change double- strandgenomic DNA by heat.
Selective enzymatic amplification of DNA can be done insmall amounts of sample.This technique is useful fordetecting a low number of parasites in stool samples . Schematic of Polymerase Chain ReactionAdvantagesof PCR:1. Extremely high sensitivity2. Easy to set upFast analysisDisadvantagesof PCRpossibility for contaminationskilled personnel is requiredQualitative result(b)Real time quantitative PCRQuantitative PCRproduct is monitored during the PCR exponential phase of reaction. it’s used commonlyin viral diagnosis , two common methods are used for pathogen detection:non-specific fluorescent dyes ( bind with any double-stranded DNA )specific DNA probes (labeled with a fluorescent reporter )advantages • Extremely sensitive • More reliable results • Precise quantification of target sequences • Faster result • Less chance of cross- contamination (c) Other Amplification Techniquesvariousalternative amplification techniques in-vitro have been developed, some of themare now available commercially. Thesealternative techniques include :· ligase chain reaction (LCR), for thedetection of chlamydia · nucleic acid sequence based amplification/isothermalamplification (NASBA).· strand displacement amplification, · branched DNA probes.
for detection ofquantification of HIV-RNA II. III. IV. V. VI. Current detection methods for virusesRecent methods in virus detection are used for research andtreatment purposes.
these methods are more important they have varioussensitivity and specificity. they divided into two groups :1- Conventional methods as :· morphology identification of virus by electron microscopy (EM).· Immuofluorescence (IF) forviral antigen detection .· Immunoenzyme (ELISA).2- Molecular methods arerapid , sensitive , include : · Nucleic acid hybridization with specific probes · amplification methods as qPCRVI. VII. Biosensors for Virus DetectionBiosensors, as diagnostic tool, used commonly for pathogen detectionand monitoring as bacteria and virus.
Advantages:· High sensitivity, selectivity · Fast analysis· Simple· Real time analysis· Miniaturization· Multi-analyte analysis· Trained personnel is not required.· Small sample volume required.Various types of biosensor available have beendeveloped recently as :§ Fluorescence.§ Lightscattering.§ Surface-enhancedRaman scattering (SERS).
§ Electrochemical.§ Quartz crystalmicrobalance (QCM).§ Microcantilevers (MCLs).§ SPR sensors.
VIII. Nanotechnology for Virus DetectionThe term nanometer refersto a unit (10 -9 meters).Nanotechnology isnewscience, has numerous of applications in medicine, industry, environment andelectronic. Medical applications of nanotechnology includenanoarrays, proteinarrays, nanopore in DNA and protein sequencing, cancer biomarker,infection diagnostic tools, nanosensors.DNA and protein microarraydetection has significant advances and strengthen in nanobiotechnology.
Nanosensors areefficient tools for biomolecular recognition, pathogenic diagnosis andenvironment monitoring. Various types ofbiosensor as waveguide used in medicalvirology field , and developed rapidly in last decades. In 1990s rapid andexpand development of diagnostic and detection techniques for pathogens areobserved as polymerase chain reaction (PCR) and its modifications,class-specific immunoglobulins (IgG/ IgM / IgA) Nanoparticles asGold NPs and quantum dots (semiconductors) have numerous of applications incancer biomarker, detection and treatment of infectious disease. New materialson Nano scale are discovered, available for design and fabricationas Crystal materials (gallium, phosphate, quartz )One of Nanotechnologyapplication is microfluidic/lab-on-a-chip. The analyte detection is quick,sensitive, and has more manipulability since NPs are used as tags or labels. ConclusionViraldiagnosis and surveillance are necessary steps in controlling the spread ofviral diseases, and they help in the deployment of appropriate therapeuticinterventions.In thepast, the commonly employed viral detection methods were either cell-culture ormolecule-level assays. Most of these assays are expensive, require special facilities, and provide a slow diagnosis.
Toavoid these limitations, biosensor-based approaches are becoming attractive,rapid identification of the presence of a virus especially after the successfulcommercialization of glucose and other biosensors. New techniques are effectivemanagement tools to be used in parallel with knowledge of the understanding thebiology of the pathogen and the ecology of the disease. Thus, these tools canbe excellent tool for providing information about pathogenicity and virulencefactors that will open up new possibilities for disease diagnosis .