About the Method

METHOD FOR DETECTING AND EVALUATING THE VITALITY OF LYMPHOTROPIC VIRUSES

The method for detecting lymphotropic viruses is intended to:

  • Increase the reliability of determining the infection of biological environments with hepatitis B virus (HBV), hepatitis C virus (HCV) or human immunodeficiency virus (HIV) which are lymphotropic;
  • Exclude false negative reactions when testing donor blood and patients for infection with HBV, HCV or HIV;
  • Detect of HBV, HCV or HIV in biological material (donated blood, plasma and other blood derivatives) when concentration of viral particles below the sensitivity threshold of the ELISA or PCR methods;
  • Assess the viability of viruses after exposure to chemical and physical factors developed and proposed by manufacturers in order to inactivate viruses in practical health care.

 

This method is unique.

 

Scope: The method of detection and evaluation of the viability of lymphotropic viruses recommended for use in diagnostic laboratories; stations for the collection of donated blood and its components; for controlling the antiviral effect of chemical and physical factors.

 

  1. INTRODUCTION

Hepatitis B viruses (HBV), hepatitis C viruses (HCV) and human immunodeficiency virus (HIV) are anthroponous viruses that infect cells of the human body only and, therefore, cause disease only in humans. Experimental models of these infections do not exist. There are also no cultured cell cultures on which the in vitro cytopathogenic properties and viability of these viruses could be adequately studied.

 

Characterization of hepatitis B, hepatitis C and HIV viruses.

Hepatitis B virus (HBV) is a DNA-containing virus, its replication occurs mainly in human hepatocytes . Hepatitis B virus also has lymphotropic properties – the ability to infect and replicate in human lymphocytes.

Hepatitis C virus (HCV) is an RNA-containing virus that has the ability to infect and replicate in both hepatocytes and human lymphocytes.

Human Immunodeficiency Virus (HIV) refers to enveloped retroviruses and is an RNA-containing virus. The main target cells for damage and replication are CD4 lymphocytes.

Therefore, a common property of HBV, HCV and HIV is their lymphotropism   – the ability to penetrate and replicate in lymphoid cells.

 

  1. BACKGROUNDS FOR CREATING A METHOD FOR DETECTING AND EVALUATING THE LIFFOTROPIC VIRUSES OF HBV, HCV AND HIV.

Research on the clinical issues, pathogenesis, treatment and diagnosis of chronic viral hepatitis, conducted by the specialists of the Laboratory research of chronic infectious processes (head. Prof. N. Gulyamov) of the Scientific Research Institute of Epidemiology, Microbiology and Infectious Diseases (SRIEMID) of the Ministry of Health of the Republic of Uzbekistan, as well as analysis Numerous scientific literature data have revealed that in chronic HBV and HCV infections, the severity of secondary immunodeficiency does not have a natural correlation with the nature and severity of -inflammatory and destructive processes in the liver tissue.

Along with this, there are isolated reports of the possibility of persistence of HBV and HCV, like HIV, in mononuclear blood cells, in particular in lymphocytes and macrophages. However, none of the researchers paid attention to evaluating this fact from the standpoint of the pathogenesis and clinic of viral hepatitis.

It was found that with HBV and HCV infections, inflammatory processes in the liver simultaneously develop with all the signs of hepatitis that follow from this. There is also a secondary immunodeficiency state, expressed in varying degrees of T-lymphopenia and B-lymphopenia, in imbalances in regulatory subpopulations of T-lymphocytes (T-helpers and T-suppressors), in a decrease in the immunoregulatory index (IRI), as well as in the dysammammoglobulinemia phenomena.  The degree and severity of immunodeficiency in this case did not have a logical relationship with the severity of the pathological process in the liver. In patients with chronic HBV and HCV infections, over time, the intensity of the progression of the pathological process in the liver tissue can vary from mild to severe, however, regardless of this, a stable and steady aggravation of secondary immunodeficiency occurs.

Inconsistencies in the degree of damage to the liver tissue and the depth of secondary immunodeficiency in various nosological forms of chronic viral hepatitis suggested that hepatitis and secondary immunodeficiency in HBV and HCV infections are combined processes, mutually aggravating, but not mutually conditioning.  That is, HBV and HCV, along with hepatotropy, also have a pronounced lymphotropic property – an unmediated property to form secondary immunodeficiency.  Moreover, differences in the clinical manifestations of liver tissue damage and the depth of immunodeficiency in HBV and HCV infections are caused by differences in the severity of the hepatotropic and lymphotropic properties of these viruses. It is precisely the differences in the severity of the hepatotropic and lymphotropic properties of the viruses that determine the differences in the pathogenesis, clinical manifestations and the nature of the effect of antiviral therapy in chronic HBV and HCV infections at different periods of the disease.

Analysis of the classical concept about the clinic and the outcomes of various diseases of viral etiology in humans revealed a pattern: a cyclical course with complete elimination of the pathogen and the outcome of recovery is naturally characteristic of diseases caused by viruses, including hepatitis A virus (HAV), which do not have specific tropism towards cells the immune system – lymphotropy.  On the other hand, the formation of a chronic, recurring and constantly progressing infectious process is naturally a characteristic feature of human diseases caused by viruses with a specific tropism towards immune cells, that is, lymphotropicity, to which HIV infection is a striking example.

Drawing an analogy between clinical and immunological parameters, clinical options, manifestations of the effect of antiviral therapy and disease outcomes in HAV, HBV and HCV infections, we have found that HAV has predominantly only hepatotropic properties.  Only in rare cases HAV gives chronicity, and only then when viral hepatitis develops against the background of already existing chronic liver pathology or secondary immunodeficiency.  However, HAV does not exhibit obvious lymphotropic properties. The absence of secondary immunodeficiency due to the direct hepatotropic action of HAV allows the immune system to realize an adequate antiviral protective reaction, which results in the elimination of HAV from the body and complete recovery. HBV and HCV, with varying degrees of hepatotropic effect, simultaneously have direct lymphotropic, i.e. secondary immunodeficiency formative effect.

It is the defeat of immune cells and the development of a secondary immunodeficiency state in HBV and HCV infections that limits the ability of the immune system to realize a full-fledged antiviral reaction of a macroorganism. The consequence of this is the formation of a chronic infectious process that resembles that of HIV infection.  The identity of the lymphotropic properties of HBV and HCV, as well as HIV, in addition to the formation of secondary immunodeficiency, is also confirmed by the commonality of their epidemiological features, transmission mechanisms, the development of concomitant opportunistic infections (frequent acute respiratory infections, acute intestinal infections) and, especially, the development of lymphogranulomatosis in various body tissues. The development of lymphogranulomatosis in various organs and tissues of the body is considered inherent specifically for viral lesions of the lymphoid cell system (Table 1, Fig. 1).

Considering the lymphotropic properties of HBV, it is not difficult to assume that HBV antigens (HBsAg), regardless of their titer in serum, can constantly and substantially in high concentrations persist in the cytoplasm of lymphoid elements. It is this phenomenon that we used to increase the reliability and eliminate false negative results in ELISA analysis.

Using an ELISA, we conducted studies to simultaneously test for the presence of HBsAg in the blood serum and the contents of lymphocytes in patients with suspected viral hepatitis. It was found that out of the total number of patients examined in 17% of cases, the average result of ELISA analysis of blood serum was false negative, since high titers of HBsAg were detected in the contents of lymphocytes of these individuals. These results, irrefutably prove the lymphotropic properties of HBV, and are of great practical importance to increase the reliability and reduce false-negative results when testing blood for the presence of HBsAg using ELISA.

So, the lymphotropic properties of HBV and HCV, including HIV, have the ability to penetrate and replicate in human lymphocytes.  Based on the above, we used lymphotropic properties – the ability of these viruses to penetrate and persist intracellularly in healthy human lymphocytes during their joint incubation in vitro to develop a method for detecting and assessing the viability of HBV, HCV and HIV.

Table 1. Comparative characteristics of the epidemiological, clinical, morphological and immunological properties of HAV, HBV, HCV and HIV.

Fig. 1. Hepatotropic and lymphotropic properties of HAV, HBV, HCV and HIV.

 

  • THE PRINCIPLE OF THE METHOD FOR DETECTING AND ASSESSING THE VIABILITY OF LYMPHOTROPIC VIRUSES HBV, HCV and HIV.

The method for detecting and assessing the viability of viruses is based on the use of lymphotropic properties – the ability of HBV, HCV or HIV to penetrate and persist in the lymphocytes of a healthy person.

Upon incubation of biological material with a suspension of lymphocytes of a healthy person, in the presence of HBV, HCV or HIV in the biological material, viruses penetrate the cytoplasm of lymphocytes. Further, the lymphocytes by centrifugation are concentrated in a small volume and destroyed by freezing.

The presented method consists of the following stages:

  1. Obtaining a suspension of lymphocytes from the blood of a healthy person;
  2. Incubating lymphocytes with the studied biological material (plasma, blood serum, etc.);
  3. Sedimenting lymphocytes by centrifugation, washing lymphocytes in physiological solution;
  4. Sedimentation of lymphocytes and dissolution in a small volume (in 500.0 μl of saline solution);
  5. Destruction of lymphocytes by freezing and thawing.

 

The content of the destroyed lymphocytes is subjected to ELISA or PCR study. Detection of viruses in the contents of lymphocytes by ELISA or PCR testifies to the presence and viability of viruses in the studied biological material.

  1. METHOD FOR DETECTING LYMPHOTROPIC VIRUSES HBV, HCV AND HIV IN LYMPHOCYTES OF PATIENTS AND BLOOD DONORS

The method allows to detect viruses in patients and blood donors even at low titers of viruses in plasma or serum.

5-6 ml of vein blood is collected from a test person in a test tube containing 2 ml of saline and 2-3 drops of heparin.

Lymphocytes are isolated from whole heparinized blood by separation in a solution of ficoll-verographin with a density gradient of d = 1.077 g / ml according to the method of F.Yu. Garib et al. (1995). During centrifugation, all blood cells, with the exception of lymphocytes, penetrate the thickness of the ficoll-verographin gradient and are located under it. Blood plasma stays above the gradient. A peculiar cloudy ring (“cloud”) is formed in the thickness of the gradient, consisting of a clean suspension of lymphocytes. This lymphocyte ring is carefully aspirated with a pipette and transferred to a clean centrifuge tube.

Next, twice washing the lymphocytes in 10.0 ml of physiological saline followed.

After the last centrifugation, the supernatant is removed.

The sediment containing lymphocytes is diluted in 500 μl of physiological saline, transferred to epindorf and placed in the freezer for 17-18 hours. With slow freezing, the lymphocyte membrane is destroyed and the contents of the cytoplasm enters the solution.

After 17-18 hours, the epindorf is taken out of the freezer and thawed at room temperature.

The contents of epindorf are analyzed using PCR or ELISA for the presence of HBV, HCV or HIV markers.

This method allows to concentrate viruses from 5-6 ml of plasma or serum to 500 μl of volume.   As a result, the titer of viral particles rises above the sensitivity threshold of the ELISA or PCR method. This helps to eliminate false negative results of ELISA or PCR when testing blood donors or patients for their infection with HBV, HCV or HIV.

 

  1. METHOD FOR DETECTING LYMPHOTROPIC VIRUSES IN BIOLOGICAL MATERIALS CONTAINING A VERY LOW TITER OF VIRUSES.

Obtaining lymphocytes of a healthy person.

Healthy volunteers using ELISA are tested for HBV, HCV and HIV infection. In tests as target cells, lymphocytes of healthy people with only negative test results are used.

To obtain a sufficient volume of lymphocytes, blood is taken from a healthy person (volunteer) in the morning on an empty stomach from the ulnar vein in an amount of 30-40 ml. Then, blood of 7-8 ml is transferred to centrifuge tubes containing 2 ml of physiological saline with 3 drops of heparin. The test tube mixes thoroughly;

The extraction of lymphocytes from whole heparinized blood is carried out by separation in a solution of ficoll-verographin with a density gradient of d = 1.077 g / ml according to the method of F.Yu. Garib et al. (1995). To do this, pour 2 ml of ficoll-verographin solution into clean centrifuge tubes, heparinized blood is carefully layered on its surface, and the tubes are centrifuged at 1500 rpm for 20 minutes. During centrifugation, all blood cells, with the exception of lymphocytes, penetrate the thickness of the ficoll-verographin gradient and are located under it. Blood plasma stays above the gradient. A peculiar cloudy ring (“cloud”) is formed in the thickness of the gradient, consisting of a clean suspension of lymphocytes. This lymphocyte ring is carefully aspirated with a pipette and transferred to a clean centrifuge tube;

Next, lymphocytes are washed three times in 10.0 ml of physiological saline followed by centrifugation.

After the last centrifugation, the supernatant is removed. Precipitation containing lymphocytes is diluted in 500 μl of saline and mixed. A suspension of lymphocytes from all tubes is poured into one tube. A suspension of lymphocytes can be stored for no more than 1 day at a temperature of + 4 ° C.

 

Incubation of lymphocytes with the tested biological material.

6-7 ml of the studied biological material (plasma, donor blood serum or others) is poured into three clean tubes, which is subjected to ELISA or PCR testing for infection with HBV, HCV or HIV.

500-600 μl of a suspension of lymphocytes of a healthy person is added to each tube, the contents of the tubes are thoroughly mixed and placed in a thermostat at 37 ° C for 6-8 hours. Every 0.5-1 hour, the contents of the tubes are shaken and mixed. During the incubation process in the presence of viruses in the studied biological medium, the latter, due to their lymphotropic properties, adhere to the membrane surface and penetrate into the cytoplasm of lymphocytes.

After incubation, the tubes are removed from the thermostat, 2 ml of physiological saline is added to them, and the mixture is mixed and centrifuged. During centrifugation, lymphocytes are deposited on the bottom of the tubes.

After centrifugation, the supernatant is removed from all the tubes, and the sediment is transferred to one tube. Next, lymphocytes are washed twice in saline.

After the last centrifugation, the supernatant is removed from the tube, the sediment containing lymphocytes is diluted with 600 μl of physiological saline and transferred to epindorph. Epindorf is placed in the freezer of the refrigerator. With slow freezing, the lymphocyte membrane is destroyed and the content of the cytoplasm enters the solution.

After 17-18 hours, the epindorf is taken out of the freezer, thawed at room temperature.

The contents of epindorf are tested using PCR or ELISA for the presence of HBV, HCV or HIV markers.

This method allows concentrating viruses from 18-20 ml of biological medium in 500 μl of volume, to increase the titer of viral particles above the sensitivity threshold of the ELISA or PCR method. This helps to eliminate false negative results of ELISA or PCR when testing biological media (plasma, serum of donated blood and others) for infection with HBV, HCV or HIV.

 

  1. METHOD FOR EVALUATING IN VITRO THE VIABILITY OF LYMPHOTROPIC VIRUSES HBV, HCV and HIV.

 

Collecting the suspension of HBV, HCV or HIV.

 

To obtain a suspension of virus blood sampling is performed from the ulnar vein from patients infected with mono-infections of HBV, HCV or HIV, with a previously known high viral load.  Virus-containing serum is isolated from whole blood.  Then, it is subjected to a quantitative PCR test to confirm the presence of HBV, HCV or HIV and determine the titer of the viruses. Virus-containing serum is stored frozen in a freezer at a temperature below – 25 ° C.

Obtaining lymphocytes of a healthy person.

Healthy volunteers using ELISA are tested for HBV, HCV and HIV infection. In tests as target cells, lymphocytes of healthy people with only negative test results are used.

To obtain a sufficient volume of lymphocytes, blood is taken from a healthy person (volunteer) in the morning on an empty stomach from the ulnar vein in an amount of 30-40 ml. Then, blood of 7-8 ml is transferred to centrifuge tubes containing 2 ml of physiological saline with 3 drops of heparin. The test tube mixes thoroughly;

The extraction of lymphocytes from whole heparinized blood is carried out by separation in a solution of ficoll-verographin with a density gradient of d = 1.077 g / ml according to the method of F.Yu. Garib et al. (1995). To do this, pour 2 ml of ficoll-verographin solution into clean centrifuge tubes, heparinized blood is carefully layered on its surface, and the tubes are centrifuged at 1500 rpm for 20 minutes. During centrifugation, all blood cells, with the exception of lymphocytes, penetrate the thickness of the ficoll-verographin gradient and are located under it. Blood plasma stays above the gradient. A peculiar cloudy ring (“cloud”) is formed in the thickness of the gradient, consisting of a clean suspension of lymphocytes. This lymphocyte ring is carefully aspirated with a pipette and transferred to a clean centrifuge tube;

Next, lymphocytes are washed three times in 10.0 ml of physiological saline followed by centrifugation.

After the last centrifugation, the supernatant is removed. Precipitation containing lymphocytes is diluted in 500 μl of saline and mixed. A suspension of lymphocytes from all tubes is poured into one tube. A suspension of lymphocytes can be stored for no more than 1 day at a temperature of + 4 ° C.

In vitro evaluation of the ability of HBV, HCV or HIV viruses to penetrate the cytoplasm of human lymphocytes.

HBV, HCV or HIV-containing plasma is removed from the freezer and thawed at room temperature.

An equal volume (300 μl) of virus-containing plasma and suspension of lymphocytes is transferred to a clean tube using an automatic pipette. The contents are mixed and put on incubation (incubation of viruses with lymphocytes in vitro) in a thermostat at a temperature of + 37 ° C for 6-8 hours. The contents of the tube are mixed by shaking every 1.5-2 hours. During incubation, only viable viruses that exhibit a cytopathogenic effect penetrate the cytoplasm of lymphocytes.

Washing off plasma from lymphocytes. Next, the tube is removed from the thermostat. 6-8 ml of physiological saline is added to it, mixed and centrifuged at 1500 rpm. Lymphocytes are deposited on the bottom of the tubes. The supernatant is removed. Next, three times washing in physiological saline and lymphocyte sedimentation is performed.

After the last centrifugation and removal of the supernatant, the suspension of lymphocytes (sediment) is diluted in 500 μl of physiological saline and transferred to epindorph.

After that, for the destruction of lymphocytes, epindorf is placed in the freezer for 17-18 hours. With slow freezing occurs the destruction of the membranes of lymphocytes.

The supernatant from epindorf is aspirated and subjected to a quantitative PCR study to identify DNA or RNA viruses that were previously contained in the blood plasma from patients in the cytoplasm of the lymphocytes.

Evaluation of the results.

A positive result of a PCR test for the presence of RNA or DNA viruses in the contents of the cytoplasm of lymphocytes indicates the preservation of the viability of viruses, that is, their ability to penetrate and persist in human lymphocytes in vitro.

A negative result of a PCR test for the presence of RNA or DNA viruses in the contents of the cytoplasm of lymphocytes indicates a loss of viability (inactivation) of viruses (after exposure to antiviral factors of a chemical or physical nature), that is, a loss of their ability to penetrate and persist in human lymphocytes in vitro.

 

  • FIELD OF APPLICATION OF THE METHOD FOR DETECTING AND EVALUATING THE VIVILITY OF LYMPHOTROPIC VIRUSES HBV, HCV and HIV.

Blood transfusion stations: to improve the reliability of detection of lymphotropic hepatitis B viruses (HBV), hepatitis C (HCV) and human immunodeficiency virus (HIV) in biological media (donated blood, plasma and other components) containing viruses in concentrations below the threshold of sensitivity of ELISA or PCR.

Diagnostic laboratories: to improve  the reliability and to eliminate false negative results when testing for the presence of HBV, HCV or HIV infection in the blood of donors or patients with virus concentrations below the sensitivity threshold of ELISA or PCR methods.

Pharmaceutical companies: to assess the viability of HBV, HCV or HIV viruses after exposure to chemical and physical antiviral factors developed and proposed by manufacturers to inactivate viruses at various sites.

The method allows to exclude errors in diagnosis when testing donor blood and its components for infection with lymphotropic viruses HBV, HCV or HIV. The method makes it possible to objectively evaluate the effects of chemical or physical factors on the viability (pathogenicity) of lymphotropic viruses.

The method for detecting and assessing the viability of lymphotropic viruses has no analogues.

This method is registered by the Ministry of Health of the Republic of Uzbekistan

Author: Gulyamov Nariman Gulyamovich, Doctor of Medical Sciences, Professor