Studying how infective a pathogen is requires biosafety laboratories for highly infectious pathogens. The process is also labor-intensive. The presence of nucleic acids alone does not define viral shedding or the infection potential of a virus. Also, the detection of viral RNA by RT-PCR does not prove that an infectious virus is present. Specific cell cultures known to be susceptible to the virus are required to study its infection potential.
Viral RNA can be detected in many viral diseases long after infectious viral particles have disappeared. The estimation of the amount of viral particles present in a sample is usually done using viral DNA or RNA copy numbers. However, it is uncertain how well copy numbers correspond to infectivity.
Several methodologies are available for diagnosis and quantification of viruses. Examples are coronaviruses' diagnoses such as MERS, SARS-CoV, and SARS-CoV-2 (COVID-19). A standard method for virus detection uses a quantitative reverse-transcription polymerase chain reaction (RT-qPCR). This approach allows detecting viral RNA in various samples, including saliva, sputum, blood, and on surfaces. RT-qPCR measures the number of copies of viral RNA per sample or specimen.
The presence of viral RNA in a sample does not necessarily mean that it contains infectious virions. Virions could be deactivated, either by environmental factors or by mutations. Therefore, researchers typically measure the "50% tissue-culture infectious dose" or TCID50. Commonly the TCID50 is determined using the read-out of an ocular or visual inspection for cytopathic effects, such as the enlargement of the infected cells.
During a TCID50 assay, serial dilutions of a virus are added onto monolayers of cells. Monitoring infected cells allow observation of cell death. The cell death is known as the cytopathic effect, or cytopathogenic effect (CPE) as a cause of structural changes in host cells due to viral invasion. The virus induces cell lysis or cell death. For accurate results, replicate cell cultures of susceptible cells combined with serial virus dilutions are studied to establish the dilution at which half the cells become infected.
For SARS-CoV-2, the presence of viral RNA in a respiratory sample may not directly correlate with a transmission of the virus or an infection. Hence, a minimum of two (2) negative tests at least 24 hours apart using PCR is recommended by WHO as one criterion for being non-infectious.
Van Doremalen et al. reported that the virus remained viable in aerosols throughout 3 hours, reducing infectious titer from 103.5 to 102. TCID50/L (for SARS-CoV-2).
According to Walsh et al., evidence suggests that the viral load in respiratory tract samples peaks around symptom onset and decreases within one to three weeks. In upper respiratory tract samples, viral RNA generally becomes undetectable about two weeks after symptom onset (14 to 15 days). Also, some patients may not be infectious for the entire period that they are SARS-CoV-2 positive. Possibly, infectivity could be related to the viral load and time since symptom onset. As noted earlier, the presence of viral RNA may not represent the transmission of the virus itself.
According to La Scola et al., patients with cycle threshold (Ct) values ≥34 were no longer contagious. Since the Ct value of a PCR reaction is the cycle number for which the fluorescence signal of a PCR product is above the background signal, the Ct values do not correlate directly with the number of viral particles or the copy number present in the sample.
Bar-on et al., who published an excellent paper with a collection of numbers for SARS-CoV-2, reported that on average infected host cells bursts and releases viral particles at 6 to 7 x 102 plaque-forming units. Could that mean that between 600 to 1,000 viral particles are needed for the infection of a host cell?
Gustafsson et al. in 2012 evaluated the use of qPCR to determine viral titers for human herpesvirus 6 (HHV-6) in cell cultures and compared them to a traditional TCID50 assessment approach. The researchers concluded that a quantitative PCR based read-out of TCID50 proved to be more robust and was also easier to interpret.
More accurate and concise research is needed before we really understand how exactly the SARS-CoV-2 virus infects cells and how many viroids are needed for infection.
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