Viruses

History

• Iwanoski demonstrated that filtered sap from diseased tobacco plants was transmissable - filterable bacterium
• Beijernick - provided evidence that the transmissable agent was not a bacterium
• Stanley (1935) - isolated the infectious agent and called it Tobacco Mosaic Virus (virus = latin for poison)
• About the same time as Stanley's discovery, the electron microscope was invented and this enabled viruses & bacteria to be studied in detail

• Viruses are the living dead and they are not cells. (See discussion <A HREF="/courses/ss12bmi/microbes_are_cells.html" on microbes are cells and organisms </A>).
• From the above, there are three possibilities on the origin of virsues:
• If they are not cells and require living cells for replication, than viruses must not have been present prior to cellular evolution and could therefore have coevolved with cells
• Were once cells but have lost all cellular functions retaining only information to replicate themselves using hosts machinary
• Viruses have evolved from plasmids (plasmids are self replicating independent DNA) or from RNA viroids (see viroids section, below). These "early" viruses did not contain genes for capsids. As viroids moved from cell to another, it picked up such genes.

General Characteristics

• Viruses (poisons) are obligate parasites, filterable, infectious particles. Demonstrated with plant sap.
• 20nm to 14000nm in size.
• Most are smaller than bacteria (eg Adeno virus)
• Vaccinia virus is the same size as the small bacteria like Mycoplasma, Rickettsia & Chlamydia
• Contain either DNA or RNA (never both) surrounded by a protein coat (capsid) and sometimes covered by an envelope.
• Obligate intracellular parasites & multiply inside living cells by using cellular synthesis machinary

Host Range

• Most viruses infect specific host cells ie are host specific. Host specificity is due to:
• specific attachment sites on the host cells called receptors
• Receptor sites for bacteriophage are found in bacterial cell walls or fimbrae or flagella
• Animal cell membranes contain receptors for animal viruses
• availability of cellular factors required for viral multiplication in the host cells.

Viral Structure

• Nucleic acid (DNA or RNA, never both)
• may be linear or circular and
• may be single or double stranded and
• may exist as a single piece or multi-segmented pieces
• The protein coat surrounding the nucleic acid is called a capsid. A capsid
• accounts for most of the mass of the viron
• structure is determined by the viral nucleic acid
• is made up of protein subunits called capsomers. Capsomers maybe composed of
• single type of protein
• several types of proteins
• arrangement of capsomers is characteristic of a particular type of virus
• Capsomers determine antigenicity of the viron
• Some capsids are surrounded by an envelope. The envelope:
• contains lipids, proteins & carbohydrates
• is derived from
• host cell membranes (can evade host defence mechanism) and / or
• may contain some proteins synthesised from viron genes and / or
• may contain some material derived from normal host cell components
• may be covered by spikes. Spikes:
• are made up of carbohydrate & protein complexes & project out into space from the surfaces of the envelope
• used for attachment to host cells
• Influenza viruses bind to RBC to form hemagglutination (diagnostic)
• Four morphological types of viruses based on the structure of the capsid and envelope geometry.
• Helical viruses -- nucleic acid is surrounded by capsids which form long hollow rods (TMV, bacteriophage M13).
• Polyhedral virsues -- capsids forming multiple (many) sides. Icosahedron is a regular polyhedron with 20 triangular faces & 12 corners (Adeno virus).
• Enveloped -- Pleomorphic to spherical shapes due to presence of an envelope.
• Enveloped helical virus - Influenza virus
• Enveloped poyhedral virus- Herpes Simplex
• Complex-- Complex stuctures in case of Lamda, T-odd &  T-even
• polyhedral capsid  + nucleic acid forms the head
• helical tail (sheath, tail fibres + pin, and baseplate)

Naming and Taxonomy

2. Family suffix -VIRIDAE.

• Picornaviridae (piccolo: Italian for very small), contains RNA
• Retroviridae (retro, latin for backward)
• Parvoviridae (parvo, latin for small)
• Togaviridae (toga or cloak)
• Arbo (Arthropod Borne Virus)
• Reoviridae (Respiratory Enteric Orphan): not linked to a known disease

4. Some 4000 viruses belonging to 71 families, 11 subfamilies and 175 genera are known (1995).

5. Taxonomy is based on a variety of criteria:

• Host specificity (infective host)
• Animal viruses
• Plant viruses
• Bacterial viruses (bacteriophages)
• Fungal viruses (mycophages)
• Algal viruses
 
• Structural morphotype
• Nucleic acid type (RNA, DNA, SS, DS, circular, segmented etc)
• Capsid symmetry

• Some examples:
• Human Immunodeficiency virus (HIV) is the species & subspecies are designated by a number eg HIV-I

Methods of cultivating viruses

Animal viruses can be cultured in a variety of ways.

• Live animals such as mice, rabbits, guinea pigs
• Animal models are used for for diagnostic purposes: observed for symptoms of disease & observed for tissue damage
• some cannot be grown in animals. The problem of studying AIDS is slow growth of virus in humans & therefore vaccine trials take a very long to test
• Simian AIDS (1986, infects green monkeys) & feline AIDS (1987, domestic cats) are HIV related virus & the disease develops within a few months, hence are good models to study growth of the virus in different tissues.
• Chimps can be infected with one sub-species of the AIDS virus, viz HIV-1 (genus lentivirus) but no symptoms are produced.
• Genetically engineered mice strains (containing genes for human T cells & human gamma globin) developed in 1990 are suseptible to AIDS infection
• Cell cultures
• Suspension cell cultures
• Adherent cell cultures
• Embryonated eggs.
• Convinient & cheap
• Virus inoculated into 7 to 10 day chick embryonated eggs into an area most appropriate for its growth
• Chorioallantoic membrane (Pox virus)
• Amniotic (Influenza virus)
• Allantoic
• yolk sac
• Obvserve for
• Embryo death
• Embryo cell damage
• Formation of pocks / lesions on the membrane
• Look for cytpathogenic effect (CPE) or plaques
• Bacterial viruses can be grown on host specific bacteria. Zones of clearing = plaques.

Viral Multiplication

The following provides an overview of these different phases citing examples of various common viruses.

(a) Adsorption and Attachment:
Attachment site of the viron specifically "connect" to a complimentory receptor sites of a host cell.

Receptor sites are an inherited trait and can vary from one individual to another. Therefore susceptibility to a viral infection (and indeed to other infections)  can vary from one person to another.

    Bacteriophage (eg lamda phage): A chance collision followed by weak chemical bonds forming between a specific appendage, end of tail fibres and receptors on the bacterial cell wall.

    Animal viruses: Attachement appendages are unlike lamda phage as these are
        distributed all over an animal viron and vary from viron to viron. The animal
        receptors are glycoproteins found on plasma membrane.
            Non-enveloped dsDNA icosahedral adenovirus: fibres at corners of
                icosahedron
            Enveloped, 8 segmented linear ssRNA Influenza virus: Hemagglutinin spikes
                attach to sialic acid of RBC plasma membrane (cause
                hemagglutination, a hallmark of family Orthomyxoviridae (Influenza virus
                A, B, C).
            Enveloped retrovirus (dsRNA) Human Immunodeficiency Virus (HIV I & II):
                causes Acquired Immune Deficiencies (AIDS): Envelope contains
                glycoprotein spikes (gp120) which attach to CD4 receptors of T cells and
                macrophages (CD4 absent in most other cells)
 
(b) Penetration:
 

• Direct the host sythesis machinary for its multiplication.
• Bacteriophage: Lysogeny or lytic cycle.
• Animal viruses: Lysogeny (eg cancer) or Budding.

Viruses in Australia

• See "Australian Contributors to Virology" Chapter 6, pp 339-420, In History of Microbiology in Australia, Australian Society For Microbiology, Frank Fenner, (Editor), Australian Print Group, Victoria.
• The Infective Horse virus: In Sept 1994, a horse trainer and 14 horses were killed due to a mystery viral illness in Brisbane. The virus was identified as equine morbillivirus, a rare virus that can be passed from animals (mammals such as cats, guinea pigs, horses) to humans. The method of transmission is respiratory (ie via nose and mouth discharges). Symptoms include breathing difficulties, very high fever and bloody foam discharge from the nose and mouth. It is unknown what the natural host is (perhaps native wild life) and how it spreads to the horses. (Sunday Mail, 20th Aug, 1995).
• Calicivirus and contrl of rabbit populations

2. Viroids are much smaller than viruses, contain RNA only and this RNA does not
     code for proteins. Discovered in 1971.

3. A plant pathologist discovered the first viroid called Potato Tuber Spindle Viroid
        (PTLV), which causes the disease Potato Tuber Spindle Disease.

4. There are 6 ways in which viroids are different to viruses:
        Contain single circular RNA of low molecular weight (300 to 400 nucleotides).
        RNA is often internally paired (3D) and is therefore protected against cellular
              enzymes
        Contain no capsids or envelopes
        There is no requirement for helper viruses
        Viroids do not produce proteins
        RNA is always copied in the host cell nucleus
        Viroids acn only be identified by nucleotide sequencing techniques

5. Viroids have so far been shown to infect plants only. Viroids like plant viruses can
        cause serious plant diseases eg potatoe spindle disease.

6. Plant cells are protected from diseases by an impermeable cell wall. Viroids like
        plant viruses enter through wounds or via other parasites eg. plant sucking
        parasites, nematodes, fungi.  Cell death is perhaps due to RNA interference in
        cell metabolism.

7. Do viroids have an old precellular origin or are they a modern day example of a
        new and extreme example of parasitism. Recently, base sequence homology has
        been shown between viroids and introns (introns also do not code for
        polypeptides) which has led to the speculation that viroids evolved from introns

2. Prions cause 8 diseases in human and animal all of which are neurological
        disorders progressive (dementing illnesses):
            Creutzfeldt-Jacob Disease (CJD) - humans
            Kuru Disease (PNG) - spread by canabilism in humans. Spread has ceased
                due to lack of this practice.
            Scrapie Disease in sheep
            Bovine Spongiform Encephalopathy (BSE) or mad cows disease

3. Prions are infectious and infective proteins and not DNA, RNA or toxins. Why?
            Ressist inactivation by heat at 90^oC.
            Insensitive to radiation  (damages DNA)
            Insensitive to nucleic acid digesting enzymes (RNAse and DNAse)
            Sensitive to protein denaturing agenst (phenol, urea)

4. There are 3 hypothesis for the infectious nature of prions:
       (a) PrP (Prion proteins) codes for normal proteins. PrP gene is located on
            chromosome 20 in humans. Prion proteins  are membrane proteins.
            Abnormal mutant prion proteins PrP^SC fold incorrectly, and form fibres or
            fibrils which stick together in cells ie they are not organised in the plasma
            membrane properly killing the cells.
 
         (b) The prion protein might contain a small but so far undetectable nucleic acid
             that directs the synthesis of the abnormal PrP^SC protein.

        (c) The infectious agent might be an unculturable or an unculture bacterium
             which can pass through filters. A Spiroplasma-like bacterium has been seen
             in brain tissues from CJD patients.

5. Transmission:
        (a) Largely unknown but it is thought that prion proteins cause normal proteins
            to fold incorrectly.
        (b) Spread to cows via scrapie infected sheep that was used to supplement cow
            feed in 1990.
        (c) Spread to humans by the consuming the milk of affected cows has been
            ruled out.
        (d) Transmitted to mice by injection of prion proteins and disease symptoms
            result.
         (e) Some of the prion diseases are spread only in families ie it is inheritable