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Report No. 003 Novel method for detecting infectious cytomegalovirus (PML method)
Report No. Novel method for detecting infectious cytomegalovirus (PML method)
003

Summary

In transplant medicine, such as bone marrow transplants and organ transplants, the process of guiding the transplanted organ to function as part of the patient's body is crucial. The human body is originally equipped with a "rejection response" that distinguishes between one’s own body and other one’s tissues and tries to eliminate them, and in order for a transplant to be successful, it is necessary to suppress this "rejection response" appropriately. For this reason, the use of immunosuppressants to suppress rejection reactions is essential at the time of transplantation. However, this is a double-edged sword, and because it weakens the patient's immune system, infections that would not be a problem for healthy people can become fatal. This is known as opportunistic infection, and cytomegalovirus (CMV) is one of these. Antiviral drugs are often administered after transplantation. However, although the probability is low, there are cases where CMV that has developed resistance to the administered drug appears, and when the therapeutic effect of the antiviral drug is low, there is a suspicion that CMV with drug resistance may have appeared. In order to differentiate between these, it is necessary to test the drug sensitivity of CMV isolated from the patient, but conventional drug sensitivity tests require a long time to isolate the virus, so they are not practical in the field of transplant medicine, and there has been a demand for a technology that can make a rapid determination.

We have applied the results of our previous basic research to establish a new method for detecting CMV infected cells, hereafter referred to as the PML method, that detects CMV in samples. This method enables specific and highly sensitive detection of CMV in samples such as cells collected from patients without the isolation of CMV and makes it possible to perform drug susceptibility testing in a short time.

What is cytomegalovirus, CMV?

CMV is a family of herpesviruses that infects humans, and like other herpesviruses, once the virus has infected, it will remain latent for life. Most people are infected with CMV in early childhood, and in most cases, the infection does not cause any symptoms. However, in the case of an unborn child with an immature immune system, CMV infection can cause serious conditions such as congenital CMV infection. In addition, even in adults, it is important to be aware of CMV retinitis, pneumonia and other serious infections that can occur when the immune system is weakened and the body becomes more susceptible to infection, e.g. AIDS, and organ transplants. The prevention and management of these so-called opportunistic infections is an important issue in transplant medicine.

What is the PML method?

It is an in vitro infection cell detection method for detecting active CMV in samples, established using our original technology [1-3].

Principle of the PML method:
PML bodies*, which are known as one of the subdomain structures within the cell nucleus, are composed of nearly 100 different proteins, including the PML protein, and are thought to be responsible for various functions. It is known that specific structural changes are caused by infection with herpesviruses, etc. For example, when a cell is infected with CMV and the viral antigen protein IE1 is synthesized, the PML body breaks down and the PML protein spreads evenly throughout the nucleus. In the PML method, this phenomenon is applied, and SE/15 cells that constantly express GFP-PML, in which the PML protein is bound to a fluorescent protein (GFP), are used.
Figure 1. Detection of infected cells using the PML method
When the PML bodies of SE/15 cells are observed under a fluorescence microscope, they appear as 5-10 bright spots in the nucleus, as shown in Figure 1-A. However, when CMV infects the cells and IE1 protein is expressed, the image shows the spread of fluorescence throughout the nucleus, as in Figure 1-B. In the PML method, the number of cells with nuclei showing this spread of fluorescence is counted as the number of positive cells (= CMV-infected cells), and the infection titer is calculated. It is possible to perform repeated culturing, observation and quantification after initial observation without the need for fixation or staining, and it is also possible to perform longitudinal analysis even with small samples such as clinical specimens.

Features of the PML method:
  1. Superior specificity, quantifiability and operability
  2. High sensitivity: Can detect even low-infectivity clinical strains of virus with high sensitivity [2-4]
  3. High correlation with disease state: Detects active CMV in samples [2-4]

Applications of the PML method



Evaluation of the efficacy of antiviral drugs:
The PML method is characterized by its high specificity, quantitative accuracy and ease of use, and it can also detect clinical strains of virus with low infectious titers with high sensitivity. When developing antiviral drugs, it can be difficult to obtain reproducible quantitative results when working with clinical strains of virus with low infectious titers. The PML method can detect positive cells with high sensitivity even in such cases, making it possible to obtain stable data.

Detection of drug-resistant viruses using the PML method:
In transplant medicine, countermeasures against CMV infection are one of the important issues. Since it is useful if drug susceptibility testing can be performed using the infectivity of the virus as an indicator without virus isolation, we attempted to detect drug-resistant viruses using the PML method. As shown in Figure 3, it was possible to distinguish between drug-sensitive and drug-resistant strains by comparing the infectious titers in the presence and absence of the drug. Furthermore, through joint research with transplant medical institutions, we have also confirmed that it is possible to detect CMV drug-resistant strains using peripheral blood mononuclear cells derived from patients [4].
Figure 2: Detection of drug-resistant viruses
Using a patient sample containing normal blood cells (nuclei displayed in white) and CMV-infected blood cells (nuclei displayed in red), the sample is cultured with SE/15 cells. In the presence of antiviral drugs, CMV infection from the infected blood cells to the adjacent SE/15 cells is suppressed, so no positive cells with altered PML bodies are observed. However, if the infecting virus strain is drug-resistant, infection could occur even in the presence of the drug, and positive cells will be produced. Therefore, it is possible to determine whether the virus is drug-resistant by comparing the number of positive cells in the presence and absence of the antiviral drug.
Figure 3: Example of detection of drug-resistant virus using the PML method
Clinical strains of CMV that had infected peripheral blood mononuclear cells were cultured in the absence (A) and presence (B) of the drug, and the positive cells at each culture day were evaluated using the PML method. In cultures without the drug, GCV, both drug-sensitive and drug-resistant strains were confirmed to be infectious (Figure 3-A), but in the presence of the GCV, only the drug-resistant strains were infectious (Figure 3-B).
The result indicates that the PML method can apply to detect drug-resistant viruses.

* PML body
Promyelocytic leukemia protein bodies, PML bodies, are dynamic nuclear protein complexes, important for gene replication, and are composed of approximately a hundred different proteins, including a PML protein. In most cells, there are more than ten PML bodies, and it is thought that it plays a variety of roles, including controlling cell aging and inhibiting apoptosis, as well as gene replication and transcription, but the details of these functions are still unknown.

References

1. Japan Patent #4299724
2. Ueno T, et al. Novel real-time monitoring system for human cytomegalovirus-infected cells in vitro that uses a green fluorescent protein-PML-expressing cell line. Antimicrob Agents Chemother, 50, 2806-13 (2006)
3. Ueno T, and Ogawa-Goto K. Use of a GFP-PML-expressing cell line as a biosensor for human cytomegalovirus infection. Methods Mol Biol, 515, 33-44 (2009)
4. Ogawa-Goto K, Ueno T, et al. Detection of active human cytomegalovirus by the promyelocytic leukemia body assay in cultures of PBMCs from patients undergoing hematopoietic stem cell transplantation. J Med Virol, 84, 479-86 (2012)
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