Clinical virology and antiviral resistance

ELISA is used to quantify antigen-specific antibodies in patient serum for disease diagnosis. Antigen from the suspected disease agent is attached to microtiter plates. The primary antibody comes from the patient's serum, which is subsequently bound by the enzyme-conjugated secondary antibody. Measuring the production of end product allows us to detect or quantify the amount of antigen-specific antibody present in the patient's serum.

The issue is the create false positive in detecting IgM because of the presence of RF (rheumatoid factor), an IgM directed to human IgG. RF is common in people affected by autoimmune disease, but in some cases, this factor can be found also in immunocompetent individuals. If I use my reference antigens and then I put patient serum with IgG against a virus and RF, the RF will bind to IgG against the virus. So, when I use a secondary antibody against IgG, I will obtain a false positive because the secondary antibody recognizes the RF. To avoid this risk, you can

Use anti-human IgM, then human IgM for the viral antigens and finally the secondary antibody labelled with enzyme.

The last generation assay for HIV is a combination of previous tests. We use a particular membrane that is able to catch both Ab and Ag, so the test is called combo test.

All the serological assays have a problem called tradeoff between sensitivity and specificity. The more a test is sensitive, the less it is specific.

The lower the rate of false negatives, the highest the sensitivity – Sensitivity = TP / (TP + FN) [calculated by testing a reference panel of positive samples]

The lower the rate of false positives, the highest the specificity – Specificity = TN / (TN + FP) [calculated by testing a reference panel of negative samples]

We have confirmatory tests for analyzing false positive/negative results, one of these is the Western blot.

Western blot detects Ab to specific Ag, rather than to virus as in the screening test. It is more specific and less prone to generate

measure by using gradient concentration of urea, if it is low a small quantity of urea is able to destroy the bond.

Antiviral antibody in the cerebrospinal fluid: if the blood-brain barrier has been saved from the infection there is no crossing of antibodies from blood to CSF, but if it is not, we can find antibodies in this fluid. HSV is commonly found in CSF.

ANTIVIRALS

Antivirals are drugs that can decrease morbidity and mortality of both chronic and acute infections, can decrease viral transmission (treatment as prevention strategy, TasP) and can also limit economic loss. Antivirals integrate well with vaccination.

There are different categories of antiviral drugs:

1. Interferons. They are not real drugs, but they are produced physiologically by our body in response to antiviral infections. They can be administered as an extra dose to strengthen antiviral immunity. Application of interferon are now very few.
2. Direct acting antiviral: drugs selectively and specifically targeting virus

function as viral enzymes. Notreally many have been approved but rate has dramatically increased in the last 20 years3.

Host targeting agents: drugs interfering with cellular functions which are essential for virusreplication. In this case there is a conflict because you are going to disturb cell activity, but there arealso some drugs that do not interfere with cell viability.

Targets for antiviral drug are essentially related to virus replication.Viruses offer much less opportunity for selective targeting of pathogen specific functions compared tobacteria. Antiviral drug development started much later than antibacterial drug development and initiallymet with limited success with. In recent years, the advent of multiple technologies for drug designdramatically boosted antiviral research

HIV was a major driver for investments in antiviral drug development. Progress with HCV drugs was evenmore dramatic, delivering an impressing number of excellent drugs in a very short time and now we are

able to eradicate it. Still, the SARS-CoV-2 pandemic shows how developing good antiviral drugs requires a considerable time.

In the last years there have been investments in antiviral drug development, following the success against HIV.

We need drugs that enter the cell to be active with an exception that is drug inhibiting attachment and penetration.

Viruses are not as well organized as bacteria to transfer drug resistance genes to different individuals; however, recombination can combine multiple resistance mutation in the same genome.

Successfully treated viral infections: herpesviruses, influenza virus, HIV, HBV and HCV.

Viruses are genetically polymorphic, there are different strains in different geographic areas and also different strains in different individuals. It is possible to find multiple variants in the same individual, they are called quasispecies and typically develop during chronic infection. Most polymorphic viruses are the most challenging for antiviral therapy because drugs