Tools that are used in field of Virology

Tools complete introduction. Virology Field.

1. New tools discovered for diagnosis and handling of viruses Presented by: Fiza Ghafoor, Ghibta Umaima Areeba Fatima, Javeria Mehmood, Farhan Ahmed

2. Table of content: Introduction CRISPR Anti viral Drugs and Therapies 3-D Cell Culture Models

3. Introduction: In the battle against viral infections, groundbreaking tools have emerged, transforming the landscape of diagnosis and treatment. These tools promise enhanced early detection, targeted treatment, and improved patient outcomes, showcasing a proactive approach to combatting viral challenges

4. CRISPR: CRISPR, an acronym for Clustered Regularly Interspaced Short Palindromic Repeats, refers to a revolutionary genetic technology that enables precise editing of DNA within living organisms. It originated from the natural defense mechanisms of bacteria and archaea against invading viruses.

5. CRISPR-Based Diagnostics: CRISPR- Cas -based diagnostic platforms: SHERLOCK (Specific High-sensitivity Enzymatic Reporter UnLOCKing) DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter)

6. Mechanism: Cas proteins to identify and cleave target nucleic acid sequences combined with reporter molecules, these systems can generate detectable signals indicating the presence of the targeted virus

7. CRISPR-Mediated Antiviral Strategies: CRISPR-Mediated Antiviral Strategies: CRISPR technology is explored as a potential antiviral strategy to combat viral infections directly. In this approach, researchers design CRISPR systems to target and cleave the viral genome, hindering the ability of the virus to replicate or causing its destruction. While this application is still in the early stages of development, it holds promise for the treatment of viral infections by directly interfering with the viral genetic material.

8. Advantages: High specificity Rapid detection Potential for multiplexing

9. Anti Viral Drug and Therapies To fight viral infections, virology researchers are always working to create novel and more effective antiviral medications and treatments. Different approaches are used in the creation of antiviral medications, and different stages of the viral life cycle are the focus of research.

10. Replication-Inhibiting Viral Agents Analogues of nucleosides and nucleotides These substances resemble RNA or DNA-building units. They trigger an early termination of replication when they are incorporated into the viral genome, which stops the virus from replicating its genetic material. Medications used to treat HIV (such as tenofovir and lamivudine) are among the examples.

11. Continue…… Polymerase Inhibitors These medications block the enzymes that viruses use to replicate their genomes. For instance, polymerase inhibitors, such as sofosbuvir , are essential in the treatment of hepatitis C.

12. Protease Inhibitors To cleave lengthy viral polypeptides into useful proteins, certain viruses need protease enzymes. By interfering with this mechanism, protease inhibitors stop the virus from maturing into mature particles. These are frequently used (e.g., darunavir , ritonavir) in the treatment of HIV.

13. Entry Inhibitors Antiviral medications that target viral entry stop the virus from adhering to or penetrating host cells. Fusion inhibitors, such as enfuvirtide for HIV, prevent the viral envelope from fusing with the host cell membrane.

14. Treatments for RNA Interference (RNAi) A natural biological process called RNA interference (RNAi) can be used to silence particular genes, including viral genes. Small RNA molecules that can target and destroy viral RNA are introduced as part of RNA interference (RNAi) therapies to prevent the spread of viruses.

15. Immune Modulators To improve the host's capacity to fight viral infections, several antiviral medications seek to modify the immune response. This includes medications that target immunological checkpoints and interferons, which activate the immune system.

16. Limitations for Antiviral Drugs and Therapies Restricted Range of Activities Host Cell Hazard Restricted Absorbency of Tissue Challenges with Intracellular Targeting Persistent and Latency Infections

17. 3-D Cell Culture Models Cells traditionally grown in 2D cultures on flat surfaces. 2D cultures lack 3D tissue complexity; 3D cell culture improves study accuracy. 3D cell culture mimics natural tissue, enhancing the study of cellular behavior and disease. Applications include drug development and investigation of infectious diseases like viruses.

18. Establishing a 3D Cell Culture Model Steps involved in establishing a 3D cell culture model are: Selection of Cells Matrix or Scaffold Selection Cell Seeding Virus Inoculation Long-Term Maintenance

19. Selection of Cells Purposeful selection of cell lines or primary cells Matching cell types to target tissue or organ Consideration of specific cell characteristics Matrix or Scaffold Selection A 3D setting is created using natural or artificial structures called matrices or scaffolds. Natural vs. artificial materials Commonly used materials (e.g., Collagen, Matrigel, alginate, synthetic polymers)

20. Cell Seeding Placing cells into a selected matrix or scaffold Encouraging cell growth and structure formation Adjusting culture conditions for optimal development Virus Inoculation Introduction to virus exposure in 3D cultures Controlled inoculation with known virus amounts Observing virus behavior, entry, replication, and spread within 3D culture

21. Long-Term Maintenance Purpose of long-term culture Observing persistent infections and latent effects Studying prolonged effects of viral exposure

22. How 3D Cell Culture Models Work in Virus Research Spatial Organization Natural arrangement of cells in 3D models mimics native tissue structures, facilitating observation of viral interactions with specific cell types. Cell-Cell Interactions Encourages natural cell interactions Study of viral entry, replication, and host defense evasion Importance for understanding viral pathogenesis

23. Observing Immune Reactions Observation of immune responses to viral infections Activation of immune cells and cytokine release Understanding host defense mechanisms Drug Testing Evaluation of antiviral drug effectiveness A realistic environment helps in drug development Predicting drug behavior in complex tissue settings

24. Observing Immune Reactions Observation of immune responses to viral infections Activation of immune cells and cytokine release Understanding host defense mechanisms Investigating Persistent Infections Exploration of long-term effects of viral infections Study of persistent infections and viral latency Understanding chronic viral diseases over time

25. Drawbacks of 3D Cell Culture Models Cost and Resource Intensiveness: Building and maintaining 3D models require more resources. Limitations in widespread adoption due to higher costs. Technical Expertise: Requires specialized knowledge in cell biology and tissue engineering. Accessibility is limited to researchers with specific skill sets. Interpretation Challenges: Interpreting 3D model results is complex due to dynamic cellular interactions. Accurate interpretation requires interdisciplinary expertise and collaboration

26. Rapid Diagnostic Tests Quick and accurate diagnosis of viral infections is made possible by the use of rapid diagnostic tests, which also help to stop the spread of infectious diseases by allowing for prompt treatment and intervention . Here are some key types of rapid diagnostic tests commonly used for viral infections : Antigen Tests Nucleic Acid Amplification Tests (NAATs) Lateral Flow Assays Serological Tests Isothermal Nucleic Acid Amplification Tests

27. Antigen Tests Principle: Antigen tests detect specific viral proteins in patient samples. Application: They are commonly used for diagnosing acute infections and are widely employed for the rapid detection of respiratory viruses, including influenza and SARS-CoV-2 (the virus that causes COVID-19). Advantages: Antigen tests are relatively simple, cost-effective, and can provide results in a short time .

28. Nucleic Acid Amplification Tests ( NAATs) Principle: NAATs amplify and detect the genetic material (DNA or RNA) of the virus. Application: While traditional PCR (polymerase chain reaction) is not rapid, newer technologies like loop-mediated isothermal amplification (LAMP) and reverse transcription LAMP (RT-LAMP) enable faster nucleic acid amplification. These tests are highly sensitive and specific. Advantages: NAATs are highly accurate and are used for diagnosing various viral infections, including HIV, hepatitis, and respiratory viruses.

29. Lateral Flow Assays (LFAs ) Principle: LFAs use the movement of fluids along a test strip to detect the presence of specific biomolecules, such as viral antigens or antibodies. Application: LFAs are commonly used for point-of-care testing and are available for various viruses, including HIV, hepatitis B and C, and certain sexually transmitted infections. Advantages: LFAs are easy to use, require minimal equipment, and can provide rapid results.

30. Serological Tests (Antibody Tests ) Principle: Serological tests detect the presence of antibodies produced by the immune system in response to a viral infection. Application: These tests are often used to determine past infections or to assess immunity. Rapid antibody tests have been widely used during the COVID-19 pandemic to identify individuals with a previous SARS-CoV-2 infection. Advantages: Serological tests are relatively simple and can provide results quickly, but they are not as effective for early diagnosis as antigen or nucleic acid tests.

31. Isothermal Nucleic Acid Amplification Tests Principle: These tests amplify genetic material at a constant temperature, eliminating the need for thermal cycling. Application: Isothermal amplification methods, such as R ecombinase P olymerase A mplification (RPA) and Helicase-Dependent A mplification (HDA), are used for rapid nucleic acid detection in various viruses. Advantages: They offer faster turnaround times compared to traditional PCR.