Introduction depend on the host for their survival

  Introduction

·       
Viral Nanotechnology provide general idea about fast developing in the field of
immunology, virology, microbiology, chemistry, physics, and mathematical
science. Its role is  by leading
researchers and practitioners, making it both 
inclusive  and essential  resources for study and research.

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·       
by increasing demand of the applications the viral
nanotechnology is quickly expanding

·       
Viral nanotechnology characterized Similarly as An note worthy
science  that concerns itself with how to
utilize  the sub-atomic modules those
unique separate science of molecular engineering only constructs. chances to
revolutionize practices in energy, biomedicine, health care, photonics,
catalysis, electronics, and public health are diverse by present potential
applications of viral technology

·       
the demand  for new
methods and techniques  in the
prevention, diagnosis, and treatment of disease is considered as reasons for
fast growth of Viral Nanotechnology. There is also great application to be used
as methods for diagnostics, including the development of diagnostic substances
and 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 immune
system protect the body against virus by generating antibodieswhich  will recognize the virus and destroy it.

The  viruses can break the
balance and cause problems, diseases when the human immune system is weak due
to multiple reasons, so called opportunist pathogen.Viruses depend on the host
for their survival and reproducing. Viruses have many important functions for
humans, plants, animals, and the environment. such as  some of them protect the host against other
infections , by transferring genes among different species. In biomedical
research, scientists use viruses to insert new genes into cells

Structure of a virus

Virus particle composed of three
main parts:

·       
Nucleic acid  ( DNA or RNA ) core of the virus

 DNA or RNA holds all of the information for
the

virus and that makes it unique and
help to multiply.

·       
Protein Coat (capsid) –covering
over the nucleic acid which  protect it.

·       
Lipid membrane (envelope) – which covers the
capsid. naked viruses haven’t envelop

Life cycle of a basic virus

lytic cycle include :

1.    
Adsorption process in which virus particle
attaches 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 and
search 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 axis
to 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, but
ssDNA in some types , connected to the protein helix by interactions between
the negatively charged nucleic acid and positive charges on the protein.

·       
Overall, the length of a helical capsid is related to the length
of the nucleic acid contained within it

·       
examples as tobacco mosaic virus

Icosahedral

Most infections viruses are icosahedral or close spherical with
chiral icosahedral symmetry. A normal icosahedron is the ideal method for
forming a close shell from sub-units.

each triangular face has 3 identical capsomeresthat give 60 for
it. Numerous viruses as rotavirus, have more than 60 capsomers and spherical
shape.Capsomeresare  surrounded by hexons
they might be made out of various proteins.

Envelope

A few types of viruses covered themselves one of the cell
layers, either the external film encompassing a contaminated host cell or
internal 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 viruses
utilize this technique

Complex

These infections have a capsid that is neither helical nor
icosahedral, and that may have additional structures, as protein tails or a
complex external layer. for example, Enterobacteria phage T4, have boggling
composition comprising of an icosahedral head 
to a helical tail, which may have a hexagonal base plate with projecting
protein tail

Virus has two types, DNA virus and RNA virus. The genome of DNA
virus is consisted of DNA, and similarly RNA is the genetic material of a RNA
virus.

 

 

 

 

 

 

 

 IV.           
Effect of Virus on human Health

 

 

Living organisms as animals,
plants, fungi, and bacteria are all subject to be infected by viral infection. However
Viruses 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.” which
is created by Immune system  produce
antibodies to protect the host against any foreign substances. Antibodies are
specific to each type of intruder, and new set of antibodies will have to be formed
for 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 adjacent
cells production of antibodies.the benefit of interferon in viral treatment is
still under research and the  mechanism
still unknown.

in case if viral infection ,
antiviral infection can be prescribed.

List of
diseases caused by viruses

    
V.           
Diagnostic Methods of viruses

 

3 categories:

(1) Direct detection

(2) Virus isolation

(3) Serology.

1. Direct Examination of Specimen
Clinical specimen examined directly for the presence of virus particles,
virus antigen , and viral nucleic acids.

·       
Electron Microscopy morphology /
immune electron microscopy

·       
Light microscopy histological
appearance – e.g. inclusion bodies

·       
Antigen detection
immunofluorescence, ELISA etc.

·       
Molecular techniques for the
direct detection of viral genomes

 2. Indirect Examination = Virus
isolation

Ø Cell Culture

·       
Cytopathic effect

·       
Haemadsorption

·       
Confirmation by neutralization,
interference

·       
Immunofluorescence etc.

Ø Eggs pocks – haemagglutination, inclusion bodies

Ø Animals disease or death confirmation by neutralization

 3. Serology

Titres increaseof antibody between acute and convalescent stages
of  infection , detection of IgM in
primary infection.

Classical Techniques

·       
COMPLEMENT FIXATION TESTS (CFT)

·       
HAEMAGGLUTINATION INHIBITION
TESTS

·       
IMMUNOFLUORESCENCE TECHNIQUES
(IF)

·       
NEUTRALIZATION TESTS

·       
SINGLE RADIAL HAEMOLYSIS

Newer Techniques

·       
RADIOIMMUNOASSAY (RIA)

·       
ENZYME LINKED IMMUNOSORBENT ASSAY
(ELISA)

·       
PARTICLE AGGLUTINATION

·       
WESTERN BLOT (WB)

1.
Virus Isolation

For
virus isolation cultured cells, eggs and laboratory animals are used. Cell
cultures more widely used for virus isolation in many laboratories.For cellculturespreparation,
tissue fragments are first dissociated, by using  trypsin or collagenase. The cell suspension is
then 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 animal
serum. After a variable slack, the cells will attach and spread on the bottom
of the container and then isolation is started.
 

Primary and Secondary Cultures

Primary
cultures are performed by replacing the fluid two or three times per week .the
cells are separated from the vessel wall by either trypsin or EDTA since the
cultures become too crowded, and the remaining portions are used to initiate
secondary cultures.

In
both primary and secondary cultures, the cells keep some of their
characteristics from which tissue they are derived.

Cell
cultures are separated into 3 types:-

1.     Primary
cells –
which are derived from animal or human tissues and can be sub-cultured only once
or twice e.g. primary monkey.

2.     Semi-continuous
diploid cells – which are prepared from human fetal tissue and can be sub-cultured
20 to 50 times e.g. human diploid fibroblasts .

3.     Continuous
cells –
originate  fromhuman cancer cells or
animal tissue e.g.

Cell
cultures vary greatly in their susceptibility to different viruses.

precautions
in cell culture :

·       
Transportation of Specimens to the
laboratory should be as soon as possible once it’s taken.

·       
 Swabs should be placed in a bottle containing
virus transport medium.

·        
 Bodily fluids and tissues should be kept in a
sterile container.

¢ 
Identification
of a specific virus grown in infected cell cultures can be performed by
neutralization of infectivity, hemadsorption inhibition, and mmunofluorescence.

 

Effects of productive viral
replication in cell culture:

·        
Cytopathic
effect(s) (CPE) as in mumps and measles
viruses

·        
Syncitia
(cell fusion)

·        
Hemadsorption

Advantages of cell culture for virus diagnosis :

·        
Relative
ease

·        
broad
spectrum

·        
Sensitivity.

Limitations:

·        
Long
period (up to 4 weeks) required for result.

·        
Susceptible
to bacterial contamination.

·        
Susceptible
to toxic substances which may be present in the specimen.

·        
Many
viruses will not grow in cell culture e.g. Hepatitis B, Diarrheal viruses,
parvovirus, papillomavirus.

·        
Difficulty
in obtaining cell cultures.

2. Electron Microscopy

It’s used for detection and
identification of virus’s morphology by EM.

Advantages:

·        
Visualization
of virus

·        
Direct
identification of virus

·        
Rapid
diagnostic tool

Disadvantages:

·        
Difficult
to analyze multiple specimens

·        
Available
of minimum number of virus (around 106 virus particles per ml
for detection)

·        
Detection
is difficult for some viruses as SRSV

·        
Expensive

·        
Highly
skilled personnel are required.

Types of EM methods;-

1.     
DirectEM:negative
staining is used, small special equipment is required ,

Samples should be concentrated prior negative
staining by different methods as differential centrifugation,ammoniumsulphate
precipitation

2.     
Immunoelectron
microscopy (IEM). High sensitivity and specificity

It’s used in the following situations:

·        
Small
number of virus particle are available.

·        
Virus
which have different morphological shapes as herpes viruses and picornaviruses

·        
In
an outbreak conditions

3.
Haemagglutination Inhibition Test

virusesdirectlyagglutinateerythrocytes by binding
to specificreceptorsites on thesurface
of theerythrocyteandthischaracteristiccan be used in detection,identificationandquantitation
of thevirus

Some virus will have ability tobind
to erythrocytes (red blood cells), causing the formation of a lattice. This characteristic
is called hemagglutination, Antibodies against the viral protein prevent virus to agglutinate
the erythrocytes , it’s known haemagglutination-inhibition test (HAI)

It’s widely used for the diagnosis
of rubella and influenza virus infections.

Advantages:

·       
Rapidin detection and identification of virus(few hours)

·       
Cheap

·       
Easily prepared

Disadvantages:

·       
Non- specific

·       
low sensitivity

 

4. ELISA (enzyme-linked
immunosorbentassay ) 

enzyme-linked
immunosorbent assay (ELISA) was developed in 1970 ,
also known as solid-phase enzyme immunoassay. It is biochemical
technique mainly used to detect the presence of specific antibody or antigen in
the sample. It has been used as marker for disease diagnosis.

Various
techniques can be used in ELISA, the most important one are:

1.     Competitive
 assay

2.     Sandwich
assay

3.     Indirect
assay

Advantages:

Very
sensitive

Quantitative

(a)  Competitive
method

§  It’s
Ag-Ab complex.

§  competitive
reaction between antigen bound with primary antibody in plate and antigen exist
in the sample.

§  highly
sensitive.

§  Intensity
 of color is inversely proportional to
the concentration of antigen present in the sample.

(b) Sandwich
method

§  Insolubilized antigen binds to the
analyte (the antibody) in the samplespecifically,

§  addition of labelled enzyme linked
second antibody that binds to primary antibody.

§  Unbound antibody-enzyme conjugates are washed
off.

(c) 
Indirect assay

§  It’s
used for detection of antibody present in the sample.

§  Primaryantibody
exist in the specimen bind specifically to the antigen

§  to
remove unbound antibodies wash the solution.

§  enzyme
will conjugate secondary antibodies.

§  color
change after addition enzyme substrate.

§  Intensity
of color direct proportional to concentration of primary antibodies.

§  Cross-reactivity
might occur

§  An
extra incubation step is required

Assay
Characteristics :

1.    
Higher sensitivity; – either by selection of antibodies with a extremely high
affinity, or by reduction of the height and variability of the background
reaction, which makes very low concentrations of analyte more readily
detectable.

2.    
Higher specificity;
– by avoiding the presence of any antibody
in the assay system with specific reactivity against non-analyte epitopes, and
by selecting combinations of monoclonal antibodies which may further increase
specificity.

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 analysis
of virus infection.

basic
principle by using a fluorescein- labelled antibody to stain specific virus
antigens present in the samples, so that the stained cells fluoresces under UV
illumination.

Direct IF, the specimen is probed directly with a specific labelled
antibody against specific virus antigen.

Indirect IF, the specimen is first probed with a
non-labelled specific antibody, followed by a labelled antibody against the
first antibody.it has extra amplification step.

§  Detection
of virus antigen can be done by direct IF or indirect IF

§  Detection
of virus antibody can be determined always by indirect IF

7. Neutralization

Antibody
which protect the body from any infection can bind with virus to loss the
infectivity.

un-neutralized
virus available may be detected by some techniques  such as CPE, haemadsorption / haemagglutination.

There
are two types of neutralization:

·       
Reversible neutralization 

Dilution
Ag-Ab complex within 30 min of the formation is defined as reversible
neutralization.

·       
Stable neutralization 

This process
is stable for long time ( several hours ) . It’s can’t be reversed by dilution.
antibodies or virion don’t change.

8. Molecular Techniques

various molecular techniques have
been developed in the last 10 years for detection microorganisms. these methods
have high sensitivity and specificity than conventional techniques.

several techniques
used in molecular detection of virus such as :

 

 

(a) Polymerase
Chain Reaction(PCR)

PCR based on change double- strand
genomic DNA by heat. Selective enzymatic amplification of DNA can be done in
small amounts of sample.

This technique is useful for
detecting a low number of parasites in stool samples .

 

               
Schematic of  Polymerase Chain Reaction

Advantages
of PCR:

1.     Extremely high sensitivity

2.     Easy to set up

Fast
analysis

Disadvantages
of PCR

possibility for contaminationskilled personnel is  requiredQualitative result

(b)
Real time quantitative PCR

Quantitative PCR
product is monitored during the PCR exponential phase of reaction. it’s used commonly
in 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 Techniques

various
alternative amplification techniques in-vitro have been developed, some of them
are now available commercially.  These
alternative techniques include :

·       
ligase chain reaction (LCR), for the
detection of chlamydia

·       
nucleic acid sequence based amplification/isothermal
amplification (NASBA).

·       
strand displacement amplification,

·       
branched DNA probes. for detection of
quantification of HIV-RNA

 
II.           
 

 
III.           
 

 
IV.           
 

 
V.           
 

  VI.           
Current detection methods for viruses

Recent methods in virus detection are used for research and
treatment purposes. these methods are more important they have various
sensitivity and specificity. they divided into two groups :

1- Conventional methods as :

·       
morphology identification of virus by electron microscopy (EM).

·       
Immuofluorescence (IF)  for
viral antigen detection .

·       
 Immunoenzyme (ELISA).

2- Molecular methods  are
rapid , sensitive , include  :

 

·       
Nucleic acid hybridization with specific probes

·       
amplification methods as qPCR

VI.           
 

VII.           
Biosensors for Virus Detection

Biosensors, as diagnostic tool, used commonly for pathogen detection
and 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 been
developed recently as :

§  Fluorescence.

§  Light
scattering.

§  Surface-enhanced
Raman scattering (SERS).

§  Electrochemical.

§  Quartz crystal
microbalance (QCM).

§  Micro
cantilevers (MCLs).

§  SPR sensors.

 

VIII.           
Nanotechnology for Virus Detection

The term nanometer refers
to a unit (10 -9 meters).

Nanotechnology is
newscience, has numerous of applications in medicine, industry, environment and
electronic. Medical applications of nanotechnology includenanoarrays, protein
arrays, nanopore in DNA and protein sequencing, cancer biomarker,
infection diagnostic tools, nanosensors.

DNA and protein microarray
detection has significant advances and strengthen in nanobiotechnology.

Nanosensors are
efficient tools for biomolecular recognition, pathogenic diagnosis and
environment monitoring.

Various types of
biosensor as waveguide used  in medical
virology field , and developed rapidly in last decades. In 1990s rapid and
expand development of diagnostic and detection techniques for pathogens are
observed as polymerase chain reaction (PCR) and its modifications,
class-specific immunoglobulins (IgG/ IgM / IgA)

 

Nanoparticles as
Gold NPs and quantum dots (semiconductors) have numerous of applications in
cancer biomarker, detection and treatment of infectious disease. New materials
on Nano scale are discovered, available for design and fabricationas  Crystal materials  (gallium, phosphate, quartz )

One of Nanotechnology
application is microfluidic/lab-on-a-chip. The analyte detection is quick,
sensitive, and has more manipulability since NPs are used as tags or labels.

 

Conclusion

Viral
diagnosis and surveillance are necessary steps in controlling the spread of
viral diseases, and they help in the deployment of appropriate therapeutic
interventions.

In the
past, the commonly employed viral detection methods were either cell-culture or
molecule-level assays. Most of these assays are 
expensive, require special facilities, and provide a slow diagnosis. To
avoid these limitations, biosensor-based approaches are becoming attractive,
rapid identification of the presence of a virus especially after the successful
commercialization of glucose and other biosensors. New techniques are effective
management tools to be used in parallel with knowledge of the understanding the
biology of the pathogen and the ecology of the disease. Thus, these tools can
be excellent tool for providing information about pathogenicity and virulence
factors that will open up new possibilities for disease diagnosis

.