Microbiology/Pathobiology 445Lecture 3, 3Apr01

Viral Structure and Diagnosis

James I. Mullins, Ph.D.Professor and Chairman

Department of Microbiologyjmullins@u.washington.edu

Spring, 2001

Slides can be downloaded (ppt) from http://ubik.microbiol.washington.edu/Index.html

Definitions:Virion - physical particle of the virus

Definition Usage:Viruses outside of cells are usually metabolically

inert. Virions consist of either DNA of RNA (constituting the genome) usually complexed with protein into a core, surrounded by a protein coat called the capsid, altogether called a nucleocapsid. The capsid is composed of identical subunits called capsomeres. It serves to protect and to ensure efficient delivery of the nucleic acid genome to new cells. Virally encoded peplomer spikes found protruding from the envelope or at the surface of a naked virion serve the critical function of receptor recognition necessary for binding and entry into susceptible cells. For many viruses, isolated viral nucleic acid is by itself infectious, albeit less so than when it is encapsidated.

VIRUSSTRUCTURECore - nucleic acid and tightly associated proteins within the virion

Capsid - protein shell around NA or coreCapsomere - protein subunit making up the capsidNucleocapsid - core and capsid

Peplomer - ("spike”)- morphological unit projecting from the envelope or surface of a naked virion

Envelope - lipid membrane found on some viruses, often derived by budding from infected cells.

VirionsVirions One the function of the outer shells of a virion is to protect the fragile

nucleic acid genome from physical, chemical, or enzymatic damage

The outer surface of the virus is also responsible for recognition of & the first interaction with the host cell

Initially, this takes the form of binding of a specific virus-attachment protein to a cellular receptor molecule

The capsid also has a role to play in initiating infection by delivering the genome in a form in which it can interact with the host cell

Capsid Symmetry &Capsid Symmetry &Virus ArchitectureVirus Architecture

Since the approximate molecular weight of a nucleotide triplet is 1000 & the average molecular weight of a single amino acid is 150, a nucleic acid can only encode a protein that is at most 15% of its own weight

Therefore, virus capsids must be made up of multiple protein molecules (subunit construction)

Helical animal virusesHelical animal viruses The simplest way to arrange multiple, identical protein subunits is to use rotational

symmetry & to arrange the irregularly shaped proteins around the circumference of a

circle to form a disk

Multiple disks can then be stacked on top of one another to form a cylinder, with the virus

genome coated by the protein shell or contained in the hollow centre of the cylinder

Helical, naked (i.e. non-enveloped) animal viruses do not exist, but the reasons are not

clear

This category includes many of the best known human pathogens, e.g. influenza virus,

mumps & measles viruses, & Rabies virus All helical animal viruses possess single-stranded, negative-sense RNA genomes

RhabdovirusRhabdovirusparticleparticle

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Icosahedral Nucleocapsids:Many viruses appear spherical by electron microscopy, but they are actually icosahedral, with the subunits of the capsid located around the vertices or face of the icosahedron. An icosahedron has 20 equilateral triangles arranged around the face of a sphere. Triangulating a dome into 20 is the best way of producing a shell of equivalently bonded identical structures. However, all known viruses have more than 20 subunits, generally 60 x N subunits

Enveloped VirusesEnveloped Viruses 'Naked' virus particles, i.e. those in which the capsid proteins are exposed to the

external environment, are produced from infected cells at the end of the replicative cycle when the cell dies, breaks down & lyses, releasing the virions.

Many viruses have devised strategies to exit from the infected cell without its total destruction.

All living cells are covered by a membrane composed of a lipid bilayer - the viability of the cell depends on the integrity of this membrane. Viruses leaving the cell must, therefore, allow this membrane to remain intact.

This is achieved by extrusion (buddingbudding) of the particle through the membrane, during which process the particle becomes coated in a lipid envelope derived from the host cell membrane & with a similar composition.

Envelope proteinsEnvelope proteins If the virus particle became covered in a smooth, unbroken lipid

bilayer, this would be its undoing. Such a coating is effectively inert, & although effective in

preventing desiccation of or enzymatic damage to the particle, would not permit recognition of receptor molecules on the host cell.

Therefore, viruses modify their lipid envelopes by the synthesis of several classes of proteins which are associated in one of three ways with the envelope.

Matrix proteinsMatrix proteins

These are internal virion proteins whose function is effectively to link the internal nucleocapsid assembly to the envelope.

Such proteins are not usually glycosylated & are often very abundant. For example, in retroviruses they comprise approximately 30%

of the total weight of the virion

Envelope and matrix proteinsEnvelope and matrix proteins

Diagnosis of Viral InfectionDiagnosis of Viral Infection

Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA.

Past Infection, i.e., immunity, is diagnosed by the detection of HAV-IgG by EIA.

HAV Infection

FecalHAV

Symptoms

0 1 2 3 4 5 6 12

Total anti-HAV

Titer ALT

IgM anti-HAV

Months after exposure

• A battery of serological tests are used for the diagnosis of acute and chronic hepatitis B infection:

HBsAg - used as a general marker of infection anti-HBc IgM - marker of acute infection anti-HBcIgG - past or chronic infection HBeAg - indicates active replication of virus and

therefore infectiveness. Anti-HBe - virus no longer replicating. However,

the patient can still be positive for HBsAg which is made by integrated HBV.

HBV-DNA -indicates active replication of the virus, more accurate than HBeAg especially in cases of escape mutants. Used mainly for monitoring response to therapy.

Symptoms

HBeAg anti-HBe

Total anti-HBc

IgM anti-HBc

anti-HBs

HBsAg

0 4 8 12 16 20 24 28 32 36 52 100Weeks after Exposure

Titer

Acute Hepatitis B Virus Infection with Recovery

IgM anti-HBc

Total anti-HBc

HBsAg

Acute (6 months)HBeAg

Chronic (Years)anti-HBe

0 4 12 24 36 52

YearsWeeks after Exposure

Titer

Progression to Chronic HBV Infection

Diagnosis of Viral InfectionDiagnosis of Viral Infection

1. Direct Examination

2. Indirect Examination (Virus Isolation)

3. Serology

Direct ExaminationDirect Examination

1. Antigen Detection immunofluorescence, ELISA etc.

2. Electron Microscopy morphology of virus particles immune electron microscopy

3. Light Microscopy histological appearance inclusion bodies

4. Molecular Methods hybridization with specific nucleic acid probes

polymerase chain reaction (PCR)

ImmunofluorescenseImmunofluorescense

HSV-infected epithelial cells from skin lesion. (Source: Virology Laboratory, Yale-New Haven Hospital)

Positive immunofluorescence test for rabies virus antigen. (Source: CDC)

ElectronmicrographsElectronmicrographs

RotavirusAdenovirus

(courtesy of Linda Stannard, University of Cape Town, S.A.)

Immune Electron MicroscopyImmune Electron Microscopy

The sensitivity and specificity of EM may be enhanced by immune electron microscopy. There are two variants:Classical Immune electron microscopy (IEM) - the sample is treated with specific anti-sera before being put up for EM. Viral particles present will be agglutinated and thus congregate together by the antibody.

Solid phase immune electron microscopy (SPIEM) - the grid is coated with specific anti-sera. Virus particles present in the sample will be absorbed onto the grid by the antibody.

Molecular MethodsMolecular Methods

Methods based on the detection of viral genome are also commonly known as molecular methods. It is often said that molecular methods is the future direction of viral diagnosis, and it is certain that the role of molecular methods will increase rapidly in the near future

Classical Molecular TechniquesClassical Molecular Techniques

Dot-blot, Southern blot, in-situ hydridization are examples of classical techniques. They depend on the use of specific DNA/RNA probes for hybridization.

The specificity of the reaction depends on the conditions used for hybridization. However, the sensitivity of these techniques is not better than conventional viral diagnostic methods.

However, since they are usually more tedious and expensive than conventional techniques, they never found widespread acceptance.

HybridizationHybridizationTechniquesTechniques

The The PolymerasePolymeraseChain Chain ReactionReaction(PCR)(PCR)

Polymerase Chain Reaction (1)Polymerase Chain Reaction (1)

PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106

and is thus an extremely sensitive technique. It is based on an enzymatic reaction involving the use of synthetic oligonucleotides

flanking the target nucleic sequence of interest. These oligonucleotides act as primers for the thermostable Taq polymerase. Repeated

cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC), annealing of primers to their complementary sequences (50oC), and primer extension (72oC) result in the exponential production of the specific target fragment.

Further sensitivity and specificity may be obtained by the nested PCR. Detection and identification of the PCR product is usually carried out by agarose gel

electrophoresis, hybridization with a specific oligonucleotide probe, restriction enzyme analysis, or DNA sequencing.

Polymerase Chain Reaction (2)Polymerase Chain Reaction (2) Advantages of PCR:

Extremely high sensitivity, may detect down to one viral genome per sample volume Easy to set up Fast turnaround time

Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay. A positive result may be difficult to interpret, especially with latent viruses such as CMV,

where any seropositive person will have virus present in their blood irrespective whether they have disease or not.

These problems are being addressed by the arrival of commercial closed systems which requires minimum handling. The use of synthetic internal competitive targets in these commercial assays

has facilitated the accurate quantification of results. However, these assays are very expensive.

Other Newer Molecular TechniquesOther Newer Molecular Techniques

Branched DNA (bDNA) is essentially a sensitive hydridization technique which involves linear amplification. Whereas exponential amplification occurs in PCR.

Therefore, the sensitivity of bDNA lies between classical amplification techniques and PCR. Other Newer molecular techniques depend on some form of amplification.

Commercial proprietary techniques such as LCR and NASBA depend on exponential amplification of the signal or the target.

Therefore, these techniques are as susceptible to contamination as PCR and share the same advantages and disadvantages.

PCR and related techniques are bound to play an increasingly important role in the diagnosis of viral infections.

DNA chips are another promising technology where it would be possible to detect a large number of viruses, their pathogenic potential, and their drug sensitivity at the same time.

MethodTargetAmplification

SignalAmplification Thermocycling Sensitivity

CommercialExamples

PCR Exponential No Yes High Roche Amplicor

LCR No Exponential Yes High Abbot LCX

NASBA Exponential No No High Organon Teknika

Qß-Replicase No Exponential No High None

Branched DNA No Linear No Medium Chiron Quantiplex

Comparison between PCR and other nucleic acid Amplification Techniques

Indirect ExaminationIndirect Examination

1. Cell Culture cytopathic effect (CPE) haemabsorption immunofluorescence

2. Eggs pocks on CAM haemagglutination inclusion bodies

3. Animals disease or death

Virus IsolationVirus Isolation

Cell Cultures are most widely used for virus isolation, there are 3 types of cell cultures:

1. Primary cells - e.g., Monkey Kidney2. Semi-continuous cells - Human embryonic kidney and skin fibroblasts3. Continuous cells - HeLa, Vero, Hep2, CEM

Primary cell culture are widely acknowledged as the best cell culture systems available since they support the widest range of viruses. However, they are very expensive and it is often difficult to obtain a reliable supply. Continuous cells are the most easy to handle but the range of viruses supported is often limited.

Cell Culture MethodsCell Culture Methods Cell culture began early in the twentieth century with whole-organ cultures,

then progressed to methods involving individual cells, either: primary cell culturesprimary cell cultures (somatic cells from an experimental animal or taken

from a human patient which can be maintained for a short period in culture), or:

immortalized cell linesimmortalized cell lines, which, given appropriate conditions, continue to grow in culture indefinitely.

Cytopathic Effect (1)Cytopathic Effect (1)

Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells . (Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town)

Cytopathic Effect (2)Cytopathic Effect (2)

Syncytium formation in cell culture caused by RSV (top), and measles virus (bottom). (courtesy of Linda Stannard, University of Cape Town, S.A.)

Plaque Plaque AssaysAssays

HaemadsorptionHaemadsorption

Syncytial formation caused by mumps virus and haemadsorption of erythrocytes onto the surface of the cell sheet. (courtesy of Linda Stannard, University of Cape Town, S.A.)

Serological MethodsSerological Methods

Complement Fixation TestComplement Fixation Test

Complement Fixation Test in Microtiter Plate. Rows 1 and 2 exhibit complement fixation obtained with acute and convalescent phase serum specimens, respectively. (2-fold serum dilutions were used) The observed 4-fold increase is significant and indicates recent infection.

ELISA for HIV antibodyELISA for HIV antibody

Microplate ELISA for HIV antibody: colored wells indicate reactivity

Western BlotWestern Blot

HIV-1 Western Blot Lane1: Positive Control Lane 2: Negative Control Sample A: Negative Sample B: Indeterminate Sample C: Positive

Animal host systems still have Animal host systems still have their uses in virology:their uses in virology:

To study viruses which cannot be propagated in vitro, e.g. HBV To study the pathogenesis of virus infections, e.g. Coxsackieviruses To test vaccine safety, e.g. oral Poliovirus vaccine.

Nevertheless, they are increasingly being discarded because: Breeding & maintenance of animals infected with viruses is expensive Whole animals are complex systems, in which it is sometimes difficult to

interpret Results obtained are not always reproducible, due to host variation Unnecessary or wasteful use of experimental animals is morally repugnant They are rapidly being overtaken by cell culture & molecular biology