Lecture notes: Human retroviruses
Retroviruses infect a wide range of animal species and cause a variety of diseases including: tumours, wasting and auto-immune diseases, immunodeficiency syndromes and aplastic and haemolytic anaemias.
They are enveloped viruses with an RNA genome. The name is derived from the fact that the virus particle contains an RNA dependent DNA Polymerase (Reverse transcriptase). This enzyme converts the RNA genome into DNA, which then integrates into the host chromosomal DNA. The reverse transcriptase is highly error prone and rapid genetic variation is a feature of this group of viruses.
The genus is divided into 5 sub genera.
Members of 2 of these genera cause disease in humans, namely Delta-retroviruses (HTLV 1 and 2) and lentiviruses (HIV 1 and 2). Genome organization
Retroviruses have a diploid genome (2 copies of RNA genome per virus particle). The genome codes for at least three genes: gag, pol and env.
LTR - gag - pol - env - (onc) - LTR
LTR - Long terminal repeat - non coding regulatory sequences at each end of the genome, which are necessary for integration into host chromosome and which also control gene expression
gag - codes for the core proteins, structural virion components pol - reverse transcriptase (polymerase) env - envelope glycoprotein onc - oncogene Oncogenes
Some retroviruses contain oncogenes. They are so called because their expression in virus-infected cells is associated with tumour development. Retroviral oncogenes are derived fromcellular genes picked up during viral integration into host DNA, way back in evolution: Most oncogenes code for proteins with growth promoting properties (such as growth factors, growth factor receptors or proteins that control the cell cycle). Their expression can lead to uncontrolled proliferation of the infected cell. This may contribute to tumour development. None of the retroviruses known to infect humans have oncogenes.
Defective virus
Many animal retroviruses are defective. Defective viruses are viruses that have lost a gene essential for replication and can therefore only undergo productive infection if the cell that is harbouring the virus is super-infected with a helper virus, which can supply the function of the lost gene.
Endogenous retroviral sequences
Integration into host DNA is a crucial step in the replication cycle of all retroviruses. This usually occurs in somatic cells. However, during their co-evolution with vertebrates, some retroviruses have integrated into germ cell DNA. This means that the retrovirus genome forms part of the genetic material of every cell and is passed down from generation to generation. A staggering eleven percent of the human genome is made up of these endogenous retroviral sequences. Fortunately they are all defective and viral replication does not occur.
 Life cycle of a typical retovirus Human retroviruses
Six human retroviruses have so far been identified. All infect T cells.
HTLV 1 - T-cell leukaemias/lymphomas, Tropical spastic paraparesis
HTLV 2 - No known pathology
HIV 1 & 2 - AIDS
Two new human retroviruses were identified recently in a few individuals from Central Africa. They are related to HTLV1 and 2 and have been called HTLV 3 and 4. No pathology has yet been attributed to them.
HTLV 1Epidemiology
HTLV1 has a world wide distribution, but there are hyperendemic foci in South West Japan, the Caribbean and parts of West Africa. In high incidence areas, up to 30% of adults may be infected. The sero-prevalence increases with age; infection is twice as common in females. Clustering of infection in families is common. Spread occurs through blood transfusion andsexual intercourse as well as mother to child transmission through breast feeding.
Clinical features
The vast majority of individuals infected by this virus harbour it asymptomatically and never develop disease. However, they may develop one (or more) of the following complications:
1. T-cell leukaemia/lymphoma. Aggressive tumour of CD4 cells which infiltrates skin and brain. Tumours are only produced after a prolonged latent period. Approximately 5% of HTLV 1 infected individuals develop this malignancy. Malignant disease is more likely to occur in individuals who acquire infection early in life.
Laboratory Diagnosis
HTLV 1 specific 1gG antibody, ELISA and western blot
HTLV1 Proviral DNA in white blood cells detected by PCR HTLV 2
This virus shares extensive nucleic acid sequence homology with HTLV 1. It was first isolated from a patient with hairy cell leukaemia, but no specific pathology has yet been attributed to it.
HIV 1 and 2Background
In the spring of 1981, a cluster of previously healthy homosexual men in New York and Los Angeles were found to be suffering from severe immunodeficiency states associated with severe opportunistic infections and rare malignancies. By 1982 the condition had been named the acquired immunodeficiency syndrome (AIDS). It was realised by this time it had an infectious aetiology because the disease could be transmitted by blood transfusions and blood products.
In 1983, a new retrovirus, termed lymphadenopathy associated virus (now called HIV 1) was isolated from the T-cells of a patient with persistent generalised lymphadenopathy.
In 1986, a second closely related virus, termed HIV 2 was isolated from a patient from West Africa with AIDS.
Currently (2010), about 34 million people are believed to be infected, world-wide; 22 million of these are in sub-Saharan Africa. HIV 1 is the major cause of the AIDS pandemic; HIV 2 is of lower virulence and infection has largely remained confined to West Africa.
Origin
AIDS is a new disease in humans. All the scientific evidence points to the disease having arisen in Africa. The reason that we think this is that HIV is very closely related to viruses that infect African monkeys, namely the simian immunodeficiency viruses (SIV). At least 10 crossings of the species barrier from monkey to man have given rise to human infections with HIV strains.
Seven crossings gave rise to HIV2 strains; one gave rise to the HIV1 group M strains. (The HIV 1 group M strains account for the current pandemic.) Two further crossings have given rise to the HIV 1 group O and N strains, respectively (groups O and N are highly divergent stains of HIV 1 which have only been found in a few individuals in the Cameroon). Another HIV 1 variant (group P) has reently been described in an individual native to the Cameroon.
HIV 2 is most closely related to an SIV strain that infects Sooty Mangabey monkeys. The virus is believed to have entered the human population in the 1940s. It is less infectious and causes a more indolent disease than HIV 1. Infection has remained largely confined to West Africa.
HIV 1 strains are most closely related to SIV strains that infect chimpanzees. In the case of the group M strains, the virus is thought to have entered the human population in the 1930s. Over the years it has evolved in its new host and diversified, giving rise to the current pandemic.
The huge diversity of HIV 1 strains in the human population is due to the high mutation rate of the virus. Strains can be grouped according to their genetic relatedness into subtypes. The different subtypes have been named according to the letters of the alphabet (A to J). Different subtypes predominate in different parts of the world. The subtype C strain of HIV 1 is the commonest subtype to be found in sub-Saharan Africa. Epidemiology - South Africa
HIV was introduced into South Africa in the 1980s. Since then the prevalence has increased enormously. Since 1990, the growth of the epidemic has been monitored by an anonymous survey of women attending ante-natal clinics in South Africa. The prevalence of HIV infection varies across the country. It is lowest in the Western Cape and highest in KwaZulu/Natal. The overall prevalence of HIV infection in adult South Africans in 2009 is estimated to be about 16.25 % (5.54 million people).
Transmission
Infection is transmitted in the same way as hepatitis B, but is much less infectious.
1.) Sexual intercourse: This is the most common route of transmission world wide. The receptive partner is at greatest risk There is an increased risk of transmission if partners have other sexually transmitted diseases and during primary HIV infection.
2.) Vertical Transmission:
In the absence of ARV prophylaxis, 10-40% of HIV-exposed babies will acquire the infection from their mothers. This is the second most common route of transmission world wide. Infection may occur in utero during birth (commonest) post-natally, through breast feeding
3.) Exposure to blood:
Intra-venous drug abusers - sharing of needles Needle-stick injuries - risk approximately 0.3% (depends on extent of the injury) muco-cutaneous exposure - risk approximately 0.1% Course of disease
HIV establishes a persistent infection in its host and only causes death many years later.
Primary infection
Most individuals experience a febrile illness about 2-4 weeks after exposure. This illness co-incides with sero-conversion (development of antibodies) and so is often referred to as the sero-conversion illness. The symptoms are similar to those of glandular fever, namely fever, sore throat, night sweats, lymphadenopathy, diarrhoea. The illness is self limiting.
Asymptomatic phase
Following the primary infection, the patient enters a stage of clinical latency. During this time the patient feels fine, but they are infectious as they have on-going viral replication. They also have HIV antibodies in their blood (and will test positive in HIV tests). This healthy state may last many years.
Prodromal phase
As the CD4 counts drop, there is a gradual onset of a variety of prodromal disorders, such as weight loss, fever, persistant lymphadenopathy, oral candidiasis and diarrhoea. These symptoms precede the progression to AIDS.
Acquired Immunodeficiency Syndrome (AIDS)Syndrome with the following features:
1) Constitutional disease: fever, diarrhoea, weight loss, skin rashes 2) Neuro-cognitave defects: dementia, myelopathy, peripheral neuropathy 3) Immunodeficiency: Increased susceptibility to opportunistic infections: 4) Rare malignancies: Kaposi sarcoma, oral hairy leukoplakia, lymphomas. Pathogenesis
When a new infection is established, the first cells to be exposed are the dendritic cells. These cells are resident in the skin and genital mucosa. It is their job to take up antigen in the tissues and to transport it to regional lymph nodes where they present it to T cells. Dendritic cells express a receptor called DC SIGN to which HIV can attach. HIV particles remain attached to the surface of the cell and are passively transported to the very cells that HIV most likes to infect, namely CD4+ T cells. Cycles of infection are set up in the CD4 cells in the lymphoid tissue.
Helper T cells are the primary target of HIV. They are cytokine secreting cells that provide the signals to control the immune response. Without them the immune response cannot function
In the early days after infection, HIV is able to replicate to very high levels while the immune system learns to deal with it. CD4 + levels in the blood fall and virus levels peak at approximately 21 days post infection. The CD4 cell population in the gut is particularly severely affected early on. However, an immune response to the virus does develop after a while and virus levels in the blood fall to a steady state level. Unfortunately, the immune response is not able to control the infection completely and virus replication continues in the lymphoid tissue. As time passes, the antiviral immunity begins to fail and virus levels begin to rise again and the person succumbs to the infection.
Deterioration is linked to the loss of CD4+ cells: Why do the T cells die?
1. Productive infection of the cell by the virus causes cell death with the release of new progeny virions.
2. Lysis of infected cells by the host's CTLs. 3. Apoptosis (activation induced cell death) of uninfected cells.
It is in the interests of the patient that the CTL killing of infected cells is efficient. If the immune system can kill the infected cells before they release new progeny viruses, virus production is less efficient and levels of virus are lower.
Thus patients with a strong CTL response have lower viral loads and survive for longer. Patients with a weak CTL response have higher viral loads and survive for a shorter time. Dying T cells are replaced by de novo synthesis of new T cells in the thymus or by cell division of mature cells in the lymphoid organs. Only when the ability of the immune system to replace dead T cells fails, do T cell numbers begin to fall.
Immune activation fuels disease progression. This is caused by ongoing virus replication and immune attack in the lymphoid tissue which damages the delicate network of immune cells. The integrity of this network is crucial for the immune system to function effectively. Once this starts to fail, cells receive incorrect signals leading to:
Inappropriate activation and death (of un-infected cells) by apoptosis Impairment of the function of the remaining cells Failure to regenerate new cells
Immune activation is made worse by the fact that the lymphoid tissue of the gut is depleted early during the clinical course of infection and the mucosal barrier to the entry of bacterial products from the gut is compromised. These products can reach the systemic lymphoid organs such as lymph nodes and spleen and induce local inflammatory responses.
Infection in infants
The source of infection is usually the mother. About one third of babies born to HIV positive mothers will be infected unless antiretroviral prophylaxis is given to mother and baby. The most risky time for transmission is during delivery, but in utero transmissions can also occur as well as post natal transmission during breast feeding. Because of their immature immune responses, about half of the infected infants do not have a phase of clinical latency, but instead develop a progressive illness and die in the first year of life. The others will experience a latent period and may survive for 5-10 years or longer. Symptoms of HIV infection in children include:
Failue to thrive, Lymphadenopathy, diarrhoeal disease, opportunistic infections, interstitial pneumonia, parotitis etc. Tuberculosis, Pneumocystis jiroveci and CMV are very common opportunistic infections that cause the death of HIV infected children in the first year of life. In South Africa HAART is started as soon as the diagnosis is made in infants. LABORATORY DIAGNOSIS and MONITORING
Serology
The mainstay of diagnosis is the detection of HIV specific antibody. IgG develops 4-6 weeks post exposure and remains detectable for life. As all individuals become chronically infected, the presence of HIV specific antibody indicates infection.
There are two situations where further tests may be necessary to confirm a diagnosis: (a) Early infection - the period after exposure before antibody becomes detectable, (sometimes termed the "window" period). (b) Infants of HIV positive mothers: all have passively acquired HIV-specific antibody, but only 10-40% are infected. This antibody may take 12 to 18 months to disappear.
In these instances, a more direct way of demonstrating the presence of HIV is necessary, namely the detection of the virus itself:
Direct detection of virus
1. Viral p24 antigen in serum - This is a useful marker of early infection. It appears in the blood 3-5 weeks post exposure and becomes detectable approximately 6 days before antibody (during the so called window period.) Once antibody appears, the p24 antigen is usually cleared.
Blood donors, source patients of needle-stick injuries and organ donors are routinely screened for both p24 Antigen as well as HIV antibody. These days many laboratories (including our own) use a combination HIV antibody/antigen test as the primary HIV screening test.
2. Detection of viral genome (proviral DNA or viral RNA) by PCR: This is a very sensitive indicator of infection. PCR becomes positive about 2 weeks after infection and remains positive throughout the course of the infection. This is the test of choice for confirming infection in infants of HIV positive mothers
3. Culture of virus from peripheral blood mononuclear cells (PBMCs). This is difficult and not routinely done.
Markers of disease progression:
These give an idea of the stage of infection and are also useful for monitoring response to antiviral drugs: CD4 count, total lymphocyte count plasma viral RNA levels DRUG THERAPY and PROPHYLAXIS
There is no cure for HIV. However, a number of anti-HIV drugs have been developed in recent years that interfere with specific steps in the virus replication cycle. Used in combination, they halt viral replication and can prolong the life of infected individuals. A regimen of at least three drugs (HAART) has to be given simultaneously to suppress HIV replication. This is because drug resistance develops very rapidly if they are used alone. Unfortunately these drugs need to be taken for life to maintain viral suppression. They also have toxic side effects and response to therapy has to be carefully monitored. Three classes of anti-retroviral drugs are used in South African public sector treatment programmes:
Nucleoside and nucleotide reverse transriptase inhibitors Non nucleoside reverse transcriptase inhibitors Protease inhibitors
See lectures on antiretroviral therapy for more details.
PROPHYLAXIS
Short courses of anti-retroviral drugs have been used effectively to prevent HIV infection following exposure:
Infants of HIV positive mothers: Various combinations of Antiretroviral drugs given to mother and baby peri-partum, have been shown to reduce transmission to the baby. Needle-stick injuries: The incidence of transmission is 0,3%; this can be reduced by 80% if AZT is administered to the exposed person within 2 hours of exposure. Combinations of 2 or 3 different drugs given for 28 days are routinely used in South Africa to prevent these transmissions. Rape - Anti-retrovirals should be given to victim for 28 days. No human studies have been done to prove their efficacy; but they have been shown to be effective in animal models. VACCINE PROSPECTS
The development of an effective vaccine for HIV is probably still some years away. The difficulty is that traditional approaches do not work. This is because the presence of HIV-specificantibody in the blood does not prevent infection. There are various reasons for this:
Nevertheless, research is on going to identify novel ways of presenting HIV antigens to the immune system. To protect an individual from HIV, it is thought that a vaccine would need to generate a potent specific cell mediated immune response at the site of entry of virus into the body, namely at the mucosal surfaces.
WRITTEB BY
KABIR HUSSAIN BAIG
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