Human Immunodeficiency Virus – AIDS


            The emergence and pandemic spread of the AIDS- Acquired Immunodeficiency Syndrome- have posed the greatest challenge to public health in modern times. It was first recognized in the United States in 1981, as a sudden outbreak of two very rare diseases – Kaposi’s sarcoma and Pneumocystis carinii pneumonia in young adults who were homosexuals or addicted to injected narcotics. They appeared to have lost their immune competence, rendering them vulnerable to fatal infections with relatively avirulent microorganisms, this condition was given the name acquired immunodeficiency syndrome. Isolation of the etiological agent was first reported in 1983 by Luc Montagnier and colleagues from Pasteur Institute, Paris. They isolated a retrovirus from a West African patient with persistent generalized lymphadenopathy and called it lymphadenopathy associated virus (LAV). In 1984 Robert Gallo and colleagues isolated a retrovirus called HTLV-3. two antigenic types of HIV– HIV-1 & HIV-2.
            Virions of the family Retroviridae possess reverse transcriptase enzyme, hence the name. This family has been divided into 3 genera - Retrovirus (HTLV-1 & HTLV-2, oncogenic viruses), Lentivirus (HIV-1 & HIV-2 causes AIDS) and Spumavirus (Human foamy virus).

Morphology – HIV is a spherical enveloped virus, about 90-120nm in size with a three layer structure. The nucleocapsid has an outer icosahedral shell and an inner coneshaped core, enclosing the ribonucleoproteins.. The genome is diploid, composed of two identical single stranded, positive sense RNA copies. In association with viral RNA is the reverse transcriptase enzyme. When the virus infects a cell, the viral RNA is transcribed by the enzyme, first into single stranded DNA and then to double stranded DNA which is integrated into the host cell chromosome. The provirus remain latent for long periods. In response to viral promoters, the provirus initiates viral replication by directing synthesis of viral RNA and other components.
            During viral replication, when the naked virus buds out through the host cell surface membrane, it acquires a lipoprotein envelope, which consists of lipid derived from the host cell membrane and virus coded glycoprotein. The major virus coded envelope proteins are the projecting knob-like spikes on the surface and the anchoring transmembrane pedicles. The 72 glycoprotein spikes constitute the major surface component of the virus of the virus which binds to the CD4 receptors on susceptible host cells. Transmembrane pedicles causes cell fusion.
Viral genes and antigens – The genome of HIV contains 3 structural genes – gag, pol, env characteristic of all retroviruses, each codes for two or more polypeptides, and other nonstructural and regulatory genes specific for the virus. The product of these genes act as antigens.
  Structural genes :
  1. The gag gene determines the core and shell of the virus. It is expressed as a precursor protein, p55. This precursor protein is cleaved into 3 proteins-p15, p18 and p24, which make up the viral core and shell. The major core antigen is p24 which can be detected in the serum during the early stages of HIV infection.
  2. The env determines the synthesis of envelope glycoprotein gp160, which is cleaved into the two envelope components- gp120 which forms the surface spikes and gp41 which is the transmembrane anchoring protein. The gp120 is the major envelope antigen.
  3. The pol gene codes for the polymerase reverse transcriptase and other viral enzymes, such as protease and endonuclease. It is expressed as a precursor protein, which is cleaved into proteins p31, p51 and p66.
  Nonstructural and regulatory genes :
  1. tat – trans activating gene enhancing the expression of all viral genes.
  2. nef – negative factor gene down regulates viral replication.
  3. rev – regulator of virus gene enhances expression of structural proteins.
  4. vif – viral infectivity factor gene influences infectivity of viral particles.
  5. vpu & vpx in HIV-1 & HIV-2 respectively, enhances maturation and release of progeny virus from cells.
  6. vpr – stimulates promoter region of the virus.
  7. LTR- long terminal repeat sequences, one at either end, containing sequences giving promoter, enhancer and integration signals.
HIV is a highly mutable virus. It exhibits frequent antigenic variations, differences in nucleotide sequences, cell tropism, growth characteristics and cytopathology. There are differences between isolates from different places or persons, even from the same person.
Resistance – HIV is thermolabile, being inactivated in 10 minutes at 600C and in seconds at 1000C. at room temperature, in dried blood it may survive upto 7 days. It withstands lyophilization. It is susceptible to common disinfectants. It can be inactivated at room temperature in 10 minutes by treatment with 50% ethanol, 35% isopropanol, 0.5% lysol, 0.5% paraformaldehyde, 0.3% hydrogen peroxide. Because of its lipid membrane envelope, it is highly susceptible to detergents, so standard washing with detergents and hot water are adequate for decontaminating clothes and household utensils. For treatment of contaminated medical instruments, 2% glutaraldehyde solution is useful.
Pathogenicity – There are three modes of transmission of HIV : sexual, parenteral, perinatal.
Of these 80% is by sexual mode of transmission. During sexual intercourse virus gets transmitted from man to man, man to woman and woman to man. There is a ten fold increased risk for HIV transmission in patients with genital ulcers caused by syphilis, chancroid and herpes and a four fold risk in gonorrhoea, chlamydial infection, trichomoniasis and vaginosis. There are evidences that some STD pathogens are more virulent in the presence of HIV related immunodeficiency. Male circumcision provides significant protection against HIV and other STD infections.
Parenteral transmission may occur through blood transfusion. When blood is collected in the window period (the interval between the time of exposure to the virus and development of detectable levels of antibodies), tests for HIV antibodies will be negative. This period varies from 1-3 months, therefore screening of blood is not 100% safe. Infection can also be transmitted by blood products like, plasma, serum and cells from HIV positives. It can also be transmitted from the donors of bone marrow, semen and organs like cornea, kidney, heart, etc. AIDS is also transmitted by sharing blood contaminated syringes, barbor’s razor, needle-stick injury.
Perinatal or vertical transmission from mother to baby  is transmitted across the placenta before birth. Some of those who do not develop this infection before birth may develop it from the genital secretions during birth and from mother’s milk after birth (very rare).
            However, the social and domestic contact with the patient does not transmit infection. Shaking hands,  hugging, travelling with an AIDS patient, sharing public telephone, toilets, cooking and eating facilities or by the bite of mosquitoes, bed bugs, donating blood does not transmit the infection. Saliva in adults contains some nonspecific inhibitory substances like fibronectins which can prevent cell to cell transfer of virus. Thus saliva is not likely a vehicle of HIV transmission.
Replication : The virus attaches via its gp120 envelope glycoprotein spike to the CD4 antigen complex which is the primary HIV receptor on T helper cells, macrophages in lungs, dermis, monocytes, glial and microglial cells of the central nervous system. T helper cells express the highest levels of CD4, hence HIV is said to be lymphotropic. The expression of CD4 is not sufficient for HIV infection, an additional membrane receptor, CD 26, which is having a protease activity, binds to another cite on gp120. both CD4 and CD 26 binding to gp120 is required for HIV infection. After binding to the receptors, the viral envelope fuses with the target cell plasma membrane, brought about by the transmembrabe gp41. Then by pinocytosis, the nucleocapsid of the virion enters into host cell. After entry, the nucleocapsid releases its RNA into the cytoplasm. The viral reverse transcriptase, acting as an RNA-dependent DNA polymerase, makes a DNA copy of the genomic RNA. The ssDNA is made double stranded by the same enzyme and now acts as a DNA-dependent DNA polymerase, ie. reverse transcription. This dsDNA moves to the nucleus and several such molecules become integrated through the action of the viral enzyme integrase, at random sites in the host cell chromosome causing a latent infection. This latency remain until events within a virus infected cell trigger activation of the provirus. Upon activation, the integrated provirus is transcribed by cellular RNA polymerase II either for the production of mRNAs which are translated into proteins or for the production of genomic RNA for insertion into progeny virus. As the viral proteins begin to assemble within the host cell, the host cell plasma membrane is modified by insertion of gp41 and gp120. the virus leaves the cell by budding, acquiring a lipoprotein envelope which contains lipid erived from the host cell membrane and glycoproteins which are virus – encoded.
            The primary pathogenic mechanism in HIV infection is the damage caused to the CD4+ T lymphocyte. The T4 cells decrease in numbers and the T4: T8 (helper: suppresser) cell ratio is reversed. Viral infection suppress the function of infected cells without causing structural damage. Infected T cells do not appear to release normal amounts of Interleukin-2, gamma interferon and other lymphokines, therefore a marked damping effect on cell mediated immune response. Humoral mechanism is also affected. Helper T cell activity is essential for optimal B cell function. The polyclonal activation of B lymphocytes leads to hypergammaglobulinemia. They are useless immunoglobulins to irrelevant antigens and are autoantibodies. They may be responsible for allergic reactions due to immune complexes. Monocyte – macrophage function is affected apparently due to lack of secretion of activating factors by the T4 lymphocytes, as a result chemotaxis, antigen presentation and intracellular killing by these cells are diminished. The activity of NK cells and cytotoxic T lymphocytes is also affected.
Clinical features – The CDC have classified the clinical course of HIV infection into following groups :
1.      Acute HIV infection – Within 2-6 weeks of infection, most patients develop low grade fever, malaise, headache, lymphadenopathy, sometimes with rash. Spontaneous resolution occurs within one month. Tests for HIV antibodies are usually negative at the onset of illness but become positive during its course, hence the infection is also known as seroconversion illness. p24 antigen can be demonstrated at the beginning of this phase.
2.      Asymptomatic or latent infection – All persons infected with HIV pass through a phase of asymptomatic period of 1- 15 years. They show positive HIV antibody tests during this phase and are infectious. In course of time the infection progresses through various stages, such as, CD4 lymphocytopenia, opportunistic infections, persistent generalized lymphadenopathy, AIDS related complex. The CD4+ T cell count decreases steadily.
3.      Persistent generalized lymphadenopathy (PGL) – presence of enlarged lymph nodes of 1cm diameter, in two or more noncontiguous extrainguinal sites and persist for at least 3 months.
4.      AIDS related complex (ARC) – This group includes patient with considerable immunodeficiency, suffering from various opportunistic infections. The typical constitutional symptoms are fatigue, unexplained fever, persistent diarrhoea and marked weight loss. The common opportunistic infections are oral candidiasis, herpes zoster, hairy cell leukoplakia, salmonellosis or TB. Generalized lymphadenopathy and splenomegaly are usually present. ARC patients are usually severely ill and many of them progress to AIDS in a few months.
5.      AIDS – is the end-stage , the irreversible breakdown of immune defence mechanisms, leaving the patient prey to progressive opportunistic infections and malignancies. Dry cough, dyspnea, fever and the characteristic pathogens are Pneumocystis carinii and M. tuberculosis. Recurrent pneumonia is an indicative of AIDS. It affects gastrointestinal system, central nervous system, skin and malignancies.
6.      Dementia – HIV may cause direct cytopathogenic damage in the central nervous system. It can cross the blood-brain barrier and cause encephalopathy leading to loss of higher infections, progressing to dementia.
7.      Pediatric AIDS – About a third to half the number of babies born to infected mothers are infected with HIV. Many of the infected children may not survive for a year. Children develop humoral immunodeficiency early, leading to recurrent bacterial infections. Failure to thrive, chronic diarrhoea, lymphadenopathy, TB are common manifestations.
Laboratory diagnosis – Laboratory tests employed for the diagnosis of HIV infection may be classified into 3 groups :
1.      Screening tests – are serological tests which are used to screen antibodies against HIV. These are of 3 types :
a.      ELISAis a highly sensitive and specific test and a standard procedure for diagnosing HIV infection, but requires expensive equipments and takes time. The antigen is obtained from HIV grown in continuous T lymphocyte cell line or by recombinant techniques. The antigen is coated on microtitre wells, the test serum is added, and if the antibody is present, it binds to the antigen. After washing away the unbound serum, antihuman immunoglobulin linked to a suitable enzyme is added, followed by a colour forming substrate. A photometrically detectable colour is formed , read by special ELISA readers. False positive reactions if rheumatoid factor or other autoantibodies present.
b.      Rapid tests – total reaction time is 30 minutes, do not require expensive equipment but are more expensive per test than ELISA.
c.       Simple tests – takes 1-2 hours, do not require expensive equipment, based on ELISA principle.  
2.      Supplemental tests – also detects antibodies against HIV.
a.      Western blot assay – highly specific and sensitive. In this assay, HIV proteins from detergent disrupted purified virions are separated according to their electrophoretic mobility and molecular weight by PAGE, then blotted on to nitrocellulose membrane . the membrane is then cut into strips. Antibodies which attach to separated viral antigens on the strip are detected  by antihuman immunoglobulin antibody to which enzyme is tagged. Enzyme substrate is subsequently added, colour change in the presence of substrate if positive. The position of the band on the strip indicates the antigen with which the antibody has reacted.
b.      Immunofluorescence test – In this test, HIV infected cells are acetone fixed on to glass slides and then reacted with test serum followed by fluorescein conjugated- antiimmunoglobulin. A positive reaction appears as apple green fluorescence of cell membrane under fluorescence microscope.
3.      Confirmatory tests – These are the tests which confirm HIV infection in an individual .
a.      Virus isolation – HIV can be isolated from patient’s peripheral blood lymphocytes by cocultivation with normal healthy donor’s lymphocytes in the presence of mitogens and IL-2, a T cell growth factor. The isolation is a  slow process taking 3-6 weeks. The presence of the virus is detected by assays for reverse transcriptase and p24 antigen in the culture fluid. Rarely the virus can be isolated from plasma, CSF, genital secretions, brain and bone marrow.
b.      Detection of p24 antigen – detected in the serum by ELISA. As the antibody response builds up antigen tests become negative. Late in infection the antigen may reappear.
c.       Detection of viral nucleic acid – detected by hybridization and PCR.
Saliva is an acceptable and often favourable alternative to serum for HIV antibody testing, it is safer than blood as it is rarely found in the saliva of HIV infection, the concentration is low in the saliva, no possible needle stick injury associated with the specimen collection.
Prophylaxis & Treatment – The prevention depends on health education, identification of sources. No specific vaccine is available because :
·          the virus mutate rapidly and possible recombination between different HIV strains.
·         virus normally enters by the mucosal route, therefore the vaccine should induce IgA antibody.
·         Virus astablishes lifelong latent infections hiding from antibody.
·         Virus infects the cells of the immune system.
    AIDS treatment include : the treatment and prophylaxis of infections, general management,immunorestorative measures, specific anti-HIV agents. Prompt diagnosis and appropriate treatment of opportunistic infections and tumors in the early stage and the patient can resume normal life in between the episodes of the disease. General management requires the understanding and cooperation of the health staff and relatives. In immunorestorative therapy, the administration of IL-2, thymic factors, leucocyte transfusion. A number of effective drugs Zidovudine (a nucleoside analogue), didanosine, lamivudine, protease inhibitors ritonavir, indinavir.



           

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