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During a viral infection, virus particles fuse with the host cell and manipulate its bio-chemical pathway to reproduce and spread. Unlike bacterial infections, there is no easy way to exterminate a virus without causing significant damage to the host. It is, therefore, important to identify the type of pathogen before proceeding to a treatment plan. However, virus testing is also crucial in several preventative aspects of healthcare and many different tests have been developed over the years to assess and identify virus particles quickly and accurately.
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Viruses are unique organisms that tread a fine line between living and non-living. If you look at how living things are classified, you will notice that viruses do not necessarily fulfil all the criteria. However, because of what happens during a successful viral infection, scientists have decided that viruses are in fact alive. Most viruses are so tiny, 17 - 1,000 nm, that they can only be seen under an electron microscope. They vary quite widely in their shape and complexity, ranging from spherical ones that look like pop-corn balls to helical, polyhedral, and even complex ones that look like the Apollo Moon Lander.
Every virus contains genetic material either in the form of DNA or RNA, and a protein coating to help protect it. Enveloped viruses also have an additional outer lipid layer that cover the protein coat. Unlike most cells, viruses do not contain any bio-chemical machinery, their survival and reproduction, therefore, depends entirely on successful infections of living cells.
Viruses enter a host organism from the environment around it. Once inside, they look for cells to infect. Different strains of viruses display compatibility with specific types of hosts and cell types, but their mode of infection remains the same.
Virus particles attach to host cells and insert genetic instructions that recruit the cells’ biochemical mechanisms to multiply. Once ready, these particles are assembled into new virions that break free and attack adjacent cells and other potential hosts.
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Viruses cause many severe illnesses and tests need to be carried out to determine suitable treatment plans or for the effective implementation of preventative measures. These tests often serve as routine procedure or may be required during medical investigations.
Identifying infectious agents - A fever or pain can manifest during countless types of infection. While a doctor is often able to speculate what is wrong by assessing these symptoms, conclusive virus tests need to be carried out to make sure the right treatment plan is administered.
Early diagnosis – As the viral lifecycle is entirely dependent on its ability to spread from one host to another, they tend to be highly contagious but clinical symptoms do not always manifest instantly. This means a carrier can unknowingly harbour the disease and spread viral particles to others for days or even weeks. If virus testing is conducted quickly after potential exposure, it is possible to take relevant measures to minimise further spreading or damage.
Verifying immunity – Individuals gain immunity against pathogens through the exposure to diseases or by way of vaccination. This acquired immunity, however, can diminish over time. Certain virus tests are used to determine the patient’s immunity status before a major medical procedure or to confirm whether a vaccine booster dose is needed.
Assessing risk of transmission to the foetus – Pregnancy is considered an immunosuppressant condition that makes both the mother and her unborn child more susceptible to infections and other problems. During pregnancy, viral infections such as Rubella and can cause several complications, ranging from spontaneous abortion, stillbirths, congenital defects to neonatal manifestations of antenatal infection. The effects vary depending on the type and timing of the infection. Thus, virus testing plays an important role in antenatal care.
Testing for neonatal infections – Antenatal exposure to certain virus particles can cause serious infections in new-born babies. Medical teams, therefore, carry out virus tests on babies with a potential risk of developing such infections.
Testing donor blood – Before a blood donation can be accepted, it is tested for several viruses that can cause severe complications in recipients, especially if they are immunocompromised. Many virus tests can produce a false-positive result even after an infection has passed but by keeping these samples out of the blood bank, we can significantly reduce the risk off further disease transmission.
Testing donor organs before transplant - Patients undergoing organ transplant are placed on immuno-suppressant medication to prevent organ rejection, so, their risk of developing a serious infection from viral exposure is much greater than others. As a result, rigorous virus testing is carried out on donor organs before it can be approved for transplant surgery.
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A number of virus testing methods have been developed and optimised throughout the course of modern medicine. These may be used in isolation or in combination to reach definite diagnoses. Tests are chosen based on several factors; a simple or inexpensive test may first be recommended to determine the presence or absence of viral particles, while more intricate methods of virus identification are applied if the first test comes back positive.
It is not always required to isolate viral fragments to identify a virus. Medical professionals can confirm certain infections by assessing the clinical symptoms or by inspecting biopsy samples. This is most commonly used for Human Papilloma Virus (HPV) tests.
HPV infects the transitional cells of the cervix. The virus is very common and usually harmless in younger girls but can lead to cervical cancer for many women later in life. Cervical cells are routinely checked for abnormalities to determine HPV infection; squamocolumnar cells are extracted from the cervix, stained with the Papanicolaou stain and observed under a light microscope. The method identifies epithelial abnormalities, metaplasia, dysplasia and other borderline changes in host cell morphologies that can be indicative of HPV activity and early onset of cervical cancer.
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Another test that does not look for virus particles, but rather viral activity, is the virus culture test. Bodily fluids or tissue samples are collected from a patient with a suspected infection. This is then added to selected cell lines - primary rhesus monkey kidney cells (RhMK) are most commonly used for this type of diagnosis. However, depending on the virus compatibility, other standard or even transgenic cell lines may be used. The test is positive when the sample induces an infection in the healthy cells. Virus culture is a slow process and can take several weeks to show a result, but it useful as it allows pathologists to easily isolate virus particles that can then be used in additional diagnostic tests.
Herpes virus culture is often used to test for the presence of Herpes Simplex Virus (HSV) in humans. Fluid from a fresh sore is added to healthy cells in the lab to test for subsequent infection. There are eight different strains of HSV that cause serious diseases in humans. Growing and isolating virus particles from a patient sample allows for subsequent tests to determine the exact strain and administer the right treatment plan.
Viral antigen detection tests are used as a diagnostic tool in early stages of a disease. During an active infection, viral antigens often develop on the surface of infected cells or are secreted into the blood stream. Using immuno-histological techniques and solid phase immunoassays, infectious viruses can be identified in both cell and blood samples.
Antigen detection tests are routinely used to identify pp65, a Cytomegalovirus (CMV) matrix antigen in peripheral blood leukocytes. Similar immunoassays, such as the ones for the detection of Human Immunodeficiency Virus (HIV) p24, have been standardised and produce accurate readings, but others tend to be less sensitive. While additional methods of viral detection may prove more precise, viral antigen detection tests have become an important tool for rapid diagnosis. Antibody Detection Test
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During a viral infection, the host organism’s immune system produces specific antibodies against viral antigens. These attach to infected cells and help the immune system tag and eliminate the pathogenic particles.
A virus antibody detection test is used to look for antibodies against a specific virus in a patient sample, usually blood or other bodily fluids. If the antibody is found, the test is considered positive and further quantitative analyses can then be performed to determine the timeline of the infection.
Enzyme-linked immunosorbent assay (ELISA) is common immunological assay used to measure and detect antibodies, antigens and proteins in biological samples. It works by systematically binding antibodies from patient samples to known viral epitopes and producing a visual signal that can be detected and quantified. This test is usually the first one to be used for viral detection in HIV infections. If antibodies to HIV are present, further tests are performed to confirm this diagnosis. Besides medicine, ELISA is also routinely used in plant pathology and biotechnology, as well as for certain quality control (QC) checks.
Indirect fluorescent antibody (IFA) assay is a sensitive, semi-quantitative, test used for the rapid detection of viral infections. Slides coated with fixed infected cells are used to bind antibodies that may be present in a patient sample. This is then visualised using fluorescent labelled secondary antibodies and a fluorescent microscope.
This test can be used for the rapid detection of the anti-rabies antibodies in cerebral spinal fluid samples and to confirm the results of a positive ELISA test for HIV.
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Viral DNA and RNA tests use precise molecular methods to detect the presence of known pathogenic nucleic acid fragments in a patient sample.
Polymerase Chain Reaction (PCR) is a nucleic acid amplification technique, in which a selected segment of the pathogenic DNA is amplified until it reaches detectable levels. Specially designed primers are used to target a small but specific part of the virus genome. If the virus DNA is present in the patient sample, the targeted segment is amplified multiple folds. The probe, tagged to the newly synthesised DNA fragments, then emits a fluorescent signal that can be detected by the PCR machine.
Unlike animal and plant cells, some viruses have evolved to use an RNA-based reproductive mechanism. For PCR to successfully detect RNA molecules, it must first be converted to complimentary DNA (cDNA). This can be done in a one- or two-step reverse transcription PCR process.
As the PCR test is designed to identify known segments of viral genomic material, as opposed to antibodies or antigens, it is extremely useful in the early detection of an infection. The window period, which is the time between the initiation of an infection and the expression of antibodies at detectable levels, for certain viral particles, like Hepatitis C Virus (HCV), is close to two months. However, using PCR, HCV can be detected as early as 1 week after an infection.
Using this technique, medical specialists can also prevent donor blood, collected during the window period from entering blood banks. Additionally, it is also used to confirm HIV diagnosis.
Sequencing is another nucleic acid based diagnostic method that is used to generate the full sequence of a virus genome. This method can be used to highlight very small differences between two strains of viruses that cannot be distinguished through other diagnostic tests. Sequencing is also useful when specific mutations in a patient sample can dictate the susceptibility to infection and alter the anti-viral therapy required.
With the advancement of science and technology, DNA and RNA based diagnostic and detection tests are becoming cheaper, faster and more automated. While many of the virus tests mentioned in this article continue to serve as quick and accessible means to disease investigation and prevention, PCR and Sequencing techniques offer the potential for early detection and a greater understanding of virus characteristics. So, it is reasonable to assume that these will becomes the primary diagnostic tools of the future.
Dutta, S. S. (2019) ‘Enzyme-linked Immunosorbent Assay (ELISA): Methodology’, News Medical, 19 December. Available at: https://www.news-medical.net/life-sciences/Enzyme-linked-Immunosorbent-Assay-(ELISA)-Methodology.aspx (Accessed: 15 April 2020)
Schad, V. A. (2019) ‘An Optimized Cell Culture System Needed for Hepatitis E Virus Subtypes’, Infectious Disease Advisor, 24 June. Available at: https://www.infectiousdiseaseadvisor.com/home/advisor-channels/hepatitis-advisor/hepatitis-e-virus-test-model/ (Accessed: 15 April 2020).