THEME: RETROVIRUSES. HUMAN IMMUNODEFICIENCY VIRUS (HIV) LABORATORY DIAGNOSTICS OF THE HIV INFECTION AND AL I. STUDENTS? INDEPENDENT STUDY PROGRAMME 1, General characteristic and classification of the retroviruses. 2. Morphology, antigen structure, and resistance of the HIV. 3. Epidemiology of the HIV infection (source of infection, route of transmission, groups with high risk of infection). 4, Pathogenesis of the HIV infection. AIDS as terminal stage of the HIV infection. 5. Laboratory diagnostics of the HIV infection: a, Presumptive diagnostics (detection of the anti-gp antibody in the test serum with ELISA); b. Confirmation of presumptive diagnose with immunoblotting (western blot) 6. Prophylaxis and therapy of the HIV infection: characteristics of the chemotherapeutic drugs. 7. AID! issociated diseases: brief review. 1. The family Retroviridae has been divided into three sub families: + Lentivirinae (includes the causative agent of the slow virus diseases) © Oncovirinae © Spumavirinae Human Immunodeficiency virus (HIV) In 1983, Luc Montangnier isolated a retrovirus from the lymph node cell of a patient with lymphadenopathy and termed this virus lymphadenopathy-associated virus (LAV) The next year, Robert Gallo’s group confirmed and extended this finding, linking this virus to the immunodeficiency syndrome (AIDS — acquired immunodeficiency syndrome The etiological agent of AIDS belongs to the lentivirus subgroup of the family Retroviridae Morphology HIV is a spherical enveloped virus with diameter 90-120 nm. It contains two identical copies of single stranded, positive sense RNA genome. There is reverse transcriptase enzyme associated with viral RNA into the nucleocapsid. The virus contains a lipoprotein envelope (consists of lipid derived from the host cell membrane and glycoprotein of virus) and nucleocapsid. The major virus coded envelope glycoproteins are the projecting spikes on the surface (spikes bind to the CD4 receptor on susceptible host cells) Major antigens of HIV Envelop antigens: * Spike antigen gp 120 (principal envelop antigen) Transmembrane pedicle protein— gp 41 Shell antigen: © Nucleocapsid protein —p 18 Core antigens: Principal core antigen — p 24 © Other core antigens — p 15; p 55 Polymerase antigens ~ p 31; p 51; p 66 Antigenic variation HIV undergoes frequent antigenic variation of core and envelope antigens. Two distinct antigenic types of HIV have been identified HIV-1 ~ represents the original isolate from America, Europe and other Western countriesHIV-2 - represents isolates from West Africa The envelope antigens of two types are different. Their core polypeptides show some cross reactivity Resistance HIV is heat labile, being inactivated at 56°C in 30 min and in seconds at 100°C, At room temperature, it may survive up to seven days Itis inactivated in 10 min by treatment with 35% isopropyl alcohol, 70% ethanol, 0,5% lysol, 0,5% sodium hypochlorite and 3% hydrogen peroxide, detergents Epidemiology There are three modes of transmission: sexual intercourse * parenteral — it may occur through blood after receiving infected blood transfusions, blood products, sharing contaminated syringes and needles * perinatal - infection may be transmitted from an infected mother to her child either transplacentally or perinatally . Pathogenesis Infection is transmitted when the virus enters the blood or tissues of a person and comes into contact with a suitable host cell (7- lymphocytes (helper). B- lymphocytes, monocytes, macrophages, glial cells, microglia, follicular dendritic cells from tonsils) Once in the cell, RNA is transcribed by reverse transcriptase into DNA (provirus) The provirus is integrated into the genome of the infected cell causing a latent infection From time to time, lytic infection is initiated and releases progeny virions to infect other cells Viral infection can suppress the function of infected cells without causing any structural damage Clinical manifestation in HIV infections are mainly due to failure of immune responses Clinical features The center for Disease Control in Atlanta, USA has classified the clinical course of HIV infection into various groups: Group 1 — Acute HIV infection. The illness is characterized by acute onset of fever, malaise, sore throat, myalgia, arthralgia, skin rash and lymphadenopathy Group 2 — Asymptomatic infection. Group 3 - Persistent generalized lymphadenopathy. This group is characterised by enlarged nodes (more than 1 em) at two or more extragenital sites for at least three months Group 4 ~ Syptomatic HIV infections. When CD4 T-lymphocyte count falls the patient may develop symptoms like fever, diarrhea, weight loss, night sweets and opportu: infections. In addition to the opportunistic infections, patient may also develop primary lymphoma and progressive multifocal leukoencephalopathy Laboratory diagnosis Laboratory diagnosis of HIV infections includes specific tests for HIV and tests for immunodeficiency. Specific tests to diagnose of the HIV infection. 1. Antigen detection — following a single massive infection, the virus antigen (p24) and reverse transcriptase may be detected in blood after about two weeks. The p24 antigen is the earliest virus marker to appear in the blood. The p24 antigen capture assay (ELISA) using anti-p24 antibody as the solid phase can be used for detection of this antigen 2. Virus isolation - patient's lymphocytes are co-cultivated with uninfected human lymphocytes in the presence of interleukin-2. Viral replication can be detected by demonstration of reverse transcriptase activity and presence of viral antigen (viru high early in infection before antibodies appear) 3. Detection of viral nucleic acid — nucleic acid can be detected by polymerase chain reaction (PCR) The most widely used is the serological method as following: ters are4, Antibody detection, There are two types of serological tests: ELISA test, particle agglutination (latex, gelatin) Supplemental test: western blot test, indirect immunofluorescence test, radio immunoprecipitation assay Laboratory diagnostics of the HIV Non-specific tests: Total and differential leukocyte count T-lymphocyte subset assays Platelet count Ig Gand Ig A levels Tests for opportunistic infections and tumour Prophylaxis No effective vaccine has yet been found out. High rate of mutation of the virus has difficulty in developing the vacc Antiretroviral therapy Antiretroviral drugs include both: Nucleoside and non-nucleoside inhibitors of enzyme reverse transcriptase Nucleotide inhibitors Viral protease inhibitors Fusion inhibitor H. — Students Practical activi 1. Toperform ELISA with te: the test, Enzyme-linked immunosorbent assay (ELISA) or the immunoenzymic test relies on the capacity of the enzyme antibody label to break down the substrate with the formation of stained products. The number of formed enzyme-antigen-antibody complexes corresponds to the intensity of substrate staining. Peroxidase and alkaline phosphatase are commonly utilized as enzymes. ELISA is distinguished by a fairly high sensitivity and rapidity of obtaining the results (within 2 hours). To perform ELISA, one should have polystyrene plates with flat-bottom wells and automatic pipettes. To quantitate the results, the spectrophotometer (a registrator of extinction at 2.492. nm wave length) is used Procedure. The first stage of ELISA is sorption of the corresponding dilution of antigen on aa solid phase for 1-2 hrs at 37 °C and 10-12 hrs at 4 °C (sensitization). Then, the wells are washed (to remove antigen which has not been adsorbed on the carrier) with tap water and washing buffer containing 0.05 per cent Twin-20 for 5 min (twice) at room temperature. After that add test serum collected from examined persons (in 0.2 ml aliquots) diluted with a phosphate-salt solution (pH 7,2) into each well. Each serum is added into one well and placed in a 37°C incubator for -3 hrs. Then wash off the antibodies which have not reacted with the antigen, Following this stage introduce 0.2-ml portions of enzyme-linked antibodies against the human immunoglobulin (antiglobulin enzyme-linked serum) and incubate the mixture at 37 °C for two hours. The unbound conjugate is washed off with buffer three times for 10 min, Put 0.1 mi of substrate (chromogen) solution into the well and allow it to stand for 30 min in the dark at room temperature. In the process of incubation in the presence of peroxidase orthophenylendiamine is stained yellow and aminosalicylic acid, brown, To stop the reaction of substrate splitting, add 0.1 ml of 1 N H:SO, (or 1 M NaOH) into the sera to reveal specific antibody. To estimate results of well The results of the reaction are read either visually or instrumentally.To acquire with immunoblotting test. To draw the scheme of this