Viral Disease Diagnosis and Serological Methods

Diagnosis of Viral Diseases

• Diagnosis involves identifying clinical signs and symptoms, detecting the virus, or detecting exposure
• Symptoms vary depending on the virus type, tropism, and pathogenicity
• Serological methods are commonly used for virus detection

Serological Methods for Diagnosis

• Vertebrate immune systems defend against viral infections; antibody production aids viral disease diagnosis
• Serological methods detect if an animal or patient was exposed to a specific virus by identifying antibodies in blood serum
• ELISA (Enzyme-linked immunosorbent assay) involves reacting serum with fixed viral antigens
• IFA (Indirect or direct immunofluorescence assay) uses infected cells fixed to microscope slides and fluorescein-labelled antibody
• Immunoblot (Western blot) detects viral antigens or antibodies to selected viral proteins using gel electrophoresis and blotting
• Virus neutralization assays involve neutralizing antibodies

ELISA

• ELISA caused a revolution in diagnostic serology due to its diversity and widespread use in immunoassay
• ELISA combines antibody specificity with spectrophotometric enzyme assay sensitivity by conjugating antibodies with an enzyme
• ELISA's popularity stems from high sensitivity, specificity, low cost, nanogram antigen detection, and automation
• ELISA uses no toxic reagents, allows visual or spectrophotometric evaluation of colour reactions, and screens many samples simultaneously
• ELISA requires a small amount of test antigen (50 μl)

Cell Culture and Immunofluorescence Microscopy

• Cell cultures are preferred for isolating viruses, though viruses can be isolated by passage in mouse brains or embryonic eggs
• Three types of cell cultures are primary cells (e.g., Monkey Kidney Cells), semi-continuous cells (e.g., Human embryonic kidney and skin fibroblasts), and continuous cells (e.g., HeLa or Vero cells)
• Primary cell cultures are widely used, supporting a wide range of viruses
• Visible effects include cytopathic effect (CPE), such as cells dying due to infection or syncytia formation
• Haemadsorption occurs when cells can stick to mammalian red blood cells
• Immunofluorescence confirms the presence of a virus, cells are fixed and probed with fluorescein-labelled antibodies
• Immunofluorescence microscopy confirms if antibodies bound to fixed cells, indicating virus presence

Western Blot

• Western blot virus diagnostic assay probes virus proteins with labelled antibodies or detects antibodies against a virus in serum
• Purified virus, virus proteins, or recombinant proteins serve as antigens
• An antibody probe molecule complexes with a target protein if the antibody's antigen-binding site can bind to an epitope
• Hybridization reactions are specific, antibodies only bind to antigenic sites with the correct 3-D shape

Virus Neutralization

• Virus neutralization assay neutralizes antibodies in a serum sample with a known amount of infectious virus
• The serum is diluted serially, mixed with the virus, and used to infect tissue cultures
• Neutralizing antibodies in the serum bind to virus particles, preventing infection
• The last dilution of sera that prevents plaque formation is the titre of neutralizing antibodies

Non-Serological Methods for Virus Detection

• Some methods do not rely on serological techniques and cannot detect exposure via serum antibodies
• Non-serological methods detect the presence of the virus

Virus Quantitation

• Virus quantitation methods include plaque assay or tissue culture infectious dose 50 (TCID50)

Polymerase Chain Reaction (PCR)

• PCR efficiently increases the number of DNA molecules
• Denaturation involves heating double-stranded DNA (dsDNA) to 94°C to separate the strands for primer access
• Annealing is the process of cooling the reaction mixture to around 50°C, enabling primers to bind to ssDNA templates
• Elongation heats the ssDNA/primer solution to 72°C with heat-stable polymerase, PCR buffer, dNTPs, and magnesium(Mg2+) molecules
• Repeating the cycle about 30 times yields over 1 million copies of the target DNA fragment
• Fluorescent techniques and rapid air thermal cycling support real-time PCR, completing analysis in 30 minutes

Haemagglutination

• Haemagglutination is the aggregation of red blood cells (RBCs) by certain viruses through virus glycoprotein interactions with surface receptors
• The reaction occurs when sufficient virus concentration forms cross-bridges between RBCs, forming a lattice-work structure at the well bottom
• Unagglutinated RBCs form a well-defined pellet
• The haemagglutination assay detects high virus concentrations and determines the highest dilution showing complete agglutination

Control of Viral Diseases:

• The three types of vaccines which prevent/cure viral diseases through vaccination or immunization are inactivated, attenuated/modified-live, and recombinant.

Inactivated Vaccines:

Viruses that have lost affectivity through irradiation or chemical treatment.

Attenuated or Modified-Live Vaccines:

Infection is caused by virus strains that become mild/avirulent through mutations. Mutations is achieved by virus passage in cultured cells or atypical host animals.

Recombinant Vaccines:

Can base themselves off of non-infectious viral components/antigens or replicating vectors/subunit vaccines. Recombinant vaccines include those based on mRNA and DNA plasmids.

Modified-live Vaccines:

• Should elicit a a strong immunological response
• Should not be too infectious for the disease
• Should not contain contaminating viruses
• Such events should not be able to back-mutate to virulence

Inactivated Vaccines:

• No proportion of the virus should survive the inactivation process, leading to infection and disease
• Should not contain contaminating viruses
• Allergic reactions to vaccines can occur

Comparison of Attenuated and Inactivated Vaccines:

• Modified-live elicit stronger quicker cell-mediated immune responses that last longer
• Inactivated vaccines, immunity is shorter lived and multiple booster vaccinations could be required

Vaccines and Vaccine Challenges

• Modern vaccines are highly efficacious and safe due to the evolution of technologies.
• Vaccinations have led to the global eradication of smallpox and rinderpest - as well as others

Features of HIV:

• The cells that are infected are the source of virus transmission and virus infection includes the integration of the viral genome into the chromosome of cells that are infected.
• The infected cells can transfer the virs by cell-to-cell contract and exist as latent
• Several independent serotypes and subtypes of HIV can be identified
• HIV infection occurs at specific sites in the host - antibody responses may not always be vigorous
• portions of HIV proteins mirror normal cellular proteins, leading to could induce autoimmune response

Ideal properties of an anti-HIV vaccine:

• Elicits that react with all HIV strains and subtypes, induces immune cytotoxic responses against virus-infected cells, and induces immune responses that recognize latently infected cells
• Will not induce antibodies that enhance HIV infection
• Safe, showing no toxic effects and long lasting

Recombinant Vaccines:

• Molecular biotechnology offers alternative approaches that should evaluated
• Cost, safety, efficiency, stability, and case of production are the key determinants of decisions.

Methods for Developing Viral Vaccines Includes:

• Protein subunit vaccines
• Viral vector-based vaccines
• Nucleic acid vaccines
• MRNA Vaccines

Nucleic Acid Vaccines:

• Immunizations are reached with DNA molecules

MRNA (mRNA) Vaccines:

• Penetrates cells of the vaccinated individual
• Viral antigens are produced from mRNA & the immune system causes response against the viral antigens

Antiviral Drugs:

• Protect against a disease, vaccines administered prior to exposure can
• An effective immune response quickly acts on infection and both virus & virus-infected cells cleared
• Some viral diseases can be treated with these antivirals despite not offering any effective immune response