Prasanna

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Types of Immunization/Vaccination

Father of Immunology is Edward Jenner. He produced the vaccine for small pox from cow pox virus. Vaccine is a substance that is introduced into the body to prevent the disease produced by certain pathogens. Vaccines consist of dead pathogens or live but attenuated (artificially weakened) organisms.

Immunization programmes and the development of new vaccines play an important role in protecting individuals against illness. Vaccination works by safely exposing individuals to a specific pathogenic microbe, artificially increasing their immunity to it.

Vaccines are made from

• Live micro-organisms that have been ‘treated’ so that they are weakened (attenuated) and are unable to cause disease.
• Dead micro-organisms.
• Some part or product of the microorganism that can produce an immune response.

Vaccine Types

Live attenuated vaccines:
These vaccines contain modified strains of a pathogen that have been weakened but are able to multiply within the body and remain antigenic enough to induce a strong immune response. Example: Oral Polio vaccine

Heterologous vaccine:
These are a group of live attenuated vaccines produced from the strains that are pathogenic in animals and not in humans. It is a vaccine that confers protective immunity against a pathogen that shares cross-reacting antigens with the microorganisms in the vaccine. Example: Cow pox virus that protects against small pox in humans.

Killed inactivated vaccines:
These groups of vaccine are produced either by killing or inactivating the bacteria or virus by chemical treatment or heat. Example; Polio virus.

Sub unit vaccine:
The antigenic determinant / epitope (the very specific part of the microbe) is used to prepare the vaccine.

DNA Vaccines:
When the genes for microbe’s antigens are introduced into the body some cells will take up the DNA. The DNA then instructs those cells to make the antigen molecules. The cells secrete the antigens and display them on their surfaces. The body’s own cells become vaccine generating factories.

Routes of Administration

• Deep subcutaneous or intramuscular route – most vaccines
• Oral route – Oral BCG vaccine
• Intradermal route – BCG vaccine
• Scarification – Small pox vaccine
• Intranasal route – Live attenuated influenza virus

Types of Immunization

Immunization is of two types:

1. Passive Immunization
2. Active Immunization

1. Passive Immunization

• Passive immunization is produced without challenging the immune system of the body. It is done by administration of serum or gamma globulins from a person who is already immunized to a non-immune person.
• Passive immunization is the administration of preformed antibodies either intravenously or intramuscularly.
• It is used to provide rapid protection in certain infections such as diphtheria or tetanus or in the event of accidental exposure to certain pathogens such as hepatitis B.
• It is also used to provide protection in immune compromised individuals.

Passive natural immunization:

Acquired from the mother before and after birth. Before birth, immunity is transferred from mother to the fetus in the form of maternal antibodies through placenta. After birth, the antibodies (Ig A) are transferred through breast milk (Table 11.2).

Table 11.2: Passive Immunization

Passive artificial immunization:

Developed by injecting previously prepared antibodies using serum from humans or animals. This type of immunity is useful for providing immediate protection against acute infections like tetanus, measles etc.

2. Active Immunization

Active immunization is the administration of vaccines containing microbial products with or without adjuvants in order to obtain long term immunological protection against the offending microbe.

At present the normal route of vaccination in most instances is either intramuscular or subcutaneous. Oral immunization is the method of choice for polio and Salmonella typhi vaccines. However, there is an increasing awareness that this route of immunization may be the best for most immunizations since nearly all infectious agents gain entrance through the mucosal surfaces.

Active natural immunization involves activation of immune system in the body to produce antibodies. It is achieved in both clinical and subclinical infections. Active artificial immunization is achieved by the administration of vaccines or toxoids.

Antigen preparations

Most vaccines consist of attenuated organisms, killed organisms, inactivated toxins, or sub cellular fragments and more recently genes for antigens in viral ‘vectors’, and DNA itself. Thus, vaccines must be capable of targeting the immune system appropriately i.e. cellular/or humoral mechanisms (Table 11.3).
Table 11.3: Antigen Preparation Used in Vaccines.

Adjuvants

Nonliving vaccines, especially those consisting of small molecules require the inclusion of agents to enhance their effectiveness.

These adjuvants include microbial, synthetic and endogenous preparations having adjuvant activity, but at present only aluminium or calcium salts are generally used in humans.

Adjuvants should enable antigens to be slowly released, preserve antigen integrity, target antigen presenting cells and induce cytotoxic lymphocytes.

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Overview of an Transplantation

Transfer of living cells, tissues or organs from one part of the body to another or from one individual to another is known as transplantation. A tissue or organ that is removed from one site and placed to another site usually in a same or different individual is called graft. The individual who provides the graft is called donor and the individual who receives the graft is called host or recipient.

If the graft is placed into its normal anatomic location, the procedure is called orthotopic transplantation. If the graft is placed in a different site it is called heterotopic transplantation. Transplantation is the only form of treatment for most end-stage organ failure.

In clinical practice, transplantation is used to overcome a functional and anatomic deficit in the recipient. Transplantation of kidneys, hearts, livers, lungs, pancreas and bone marrow are widely done today.

Methods of Transplantation

Auto grafting:
The transfer of self tissue from one body site to another in the same individual

Allografting:
The transfer of organs or tissues from human to human

Xenografting:
The transfer of tissue from one species to another (Figure 11.11).

Graft Acceptance

When transplantation is made between genetically identical individuals the graft survives and lives as healthy as it is in the original places. When the graft tissue remains alive, it is said to be accepted and the process is called graft acceptance.

Graft Rejection

When transplantation is made between genetically distinct individual the graft tissue dies and decays. When the graft tissue dies, the graft is said to be rejected and the process is called graft rejection. It is of two types. They are:-

1. Host Verses Graft Reaction
2. Graft Verses Host Rejection.

Host Verses Graft Reaction (HVG)

The graft tissue antigens induce an immune response in the host. This type of immune response is called host versus graft reaction.

Allograft Rejection

Types of allograft rejection

• Acute rejection-Quick graft rejection. It is due to stimulation of thymocytes and B lymphocytes
• Hyperacute rejection-It is a very quick rejection. It is due to pre-existing humoral antibodies in the serum of the host as a result of presensitization with previous grafts.
• Insidious rejection-It is a secret rejection due to deposition of immune complex on the tissues like glomerulus membrane that can be demonstrated in kidney by immune fluorescence.

Mechanism of Allograft Rejection

Immunological contact

When tissue is implanted as graft, its antigen can pass into local lymph nodes of the host. The graft antigens then make contact with the lymphocytes of the host. Production of sensitized T cells and cytotoxic antibodies are produced in the host. This brings about graft rejection.

First set rejection

When the graft is made between genetically different individuals, the graft gets blood supply from the host and it appears to be normal for the first 3 days. But on the 5th day, sensitized T cells, macrophages and a few plasma cells invade the graft. Inflammation starts in the graft. This leads to necrosis. It is similar to the primary immune response to an antigen.

Second set rejection

When a graft is implanted in an individual who has already rejected a graft is second set rejection. This is similar to the secondary immune response of our body.

Cell mediated cytotoxic reaction

The 1st set of rejection of allograft is brought about mainly by CMI response. In this process the cells involved in the cytotoxic mediated immunity involves. On stimulation of these cells interferon causes the lysis of the graft.

Antibody mediated cytotoxic reaction

The 2^2nd set rejection of graft is brought about mainly by HMI response. This is one of the hyperacute rejection brought about by the antibodies. Complement, macrophages, mast cells, platelets, B cells bring about this reaction.

Graft versus Host Rejection (GVH)

Sometimes the graft tissue elicits an immune response against the host antigens. This immune response is called graft versus host reaction. It occurs when:

• Graft remains inside the host and the host should not reject the graft.
• The graft should have immune competent T cells.
• The transplantation antigens of the host should be different from that of the graft.

Mechanism of the graft rejection

The graft lymphocytes aggregate in the host lymphoid organs and are stimulated by the lymphocytes of the host. The stimulated lymphocytes produce lymphokines. Lymphocytes in turn activate the host T cell. Activated T cell further activates the B cells. The stimulated B cell reacts with the self antigen and causes the damage.

How to prevent graft rejection?

Before transplantation the following things should be done to avoid graft rejection.

• Perform blood grouping and Rh grouping
• HLA typing should be done
• Immuno suppressive drugs should be administered
• Suitable donor should be chosen

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Hypersensitivity Types and its Classification

Hypersensitivity is defined as the exaggerated immunological response leading to severe symptoms and even death in a sensitized individual when exposed for the second time. It is commonly termed as allergy. The substances causing allergic/hypersensitivity is known as allergens. Example: Drugs, food stuffs, infectious microorganisms, blood transfusion and contact chemicals.

Classification of Hypersensitivity (Coombs and Gell Classification)

Type I:
Immediate (Atopic or anaphylactic) Hypersensitivity

Type II:
Antibody-dependent Hypersensitivity

Type III:
Immune complex mediated Hypersensitivity

Type IV:
Cell mediated or delayed Hypersensitivity

Type I:
Immediate (Atopic or anaphylactic) Hypersensitivity

This type of hypersensitivity is an allergic reaction provoked by the re-exposure to a specific antigen. The antigen can make its entry through ingestion, inhalation, injection or direct contact. The reaction may involve skin, eyes, nasopharynx and gastrointestinal tract. The reaction is mediated by IgE antibodies (Figure 11.7).

IgE has very high affinity for its receptor on mast cells and basophils. Cross linking of IgE receptor is important in mast cell trigerring. Mast cell degranulation is preceded by increased Ca⁺⁺ influx.

Basophils and mast cells release pharmacologically active substances such as histamines and tryptase. This causes inflammatory response. The response is immediate (within seconds to minutes). Hence, it is termed as immediate hypersensitivity. The reaction is either local or systemic.

Hay Fever

Allergic rhinitis is commonly known as hay fever. Allergic rhinitis develops when the body’s immune system becomes sensitized and overreacts to something in the environment like pollen grains, strong odour of perfumes, dust etc that typically causes no problem in most people. When a sensitive person inhales an allergen the body’s immune system may react with the symptoms such as sneezing, cough and
puffy swollen eyelids.

Type II Hypersensitivity: Antibody dependent hypersensitivity

In this type of hypersensitivity reactions the antibodies produced by the immune response binds to antigens on the patient’s own cell surfaces. It is also known as cytotoxic hypersensitivity and may affect variety of organs or tissues. Ig G and Ig M antibodies bind to these antigens and form complexes. This inturn activates the classical complement pathway and eliminates the cells presenting the foreign antigen. The reaction takes hours to day (Figure 11.8).

Drug induced haemolytic anaemia Certain drugs such as penicillin, cephalosporin and streptomycin can absorb non-specifically to protein on surface of RBC forming complex similar to hapten-carrier complex. In some patients these complex induce formation of antibodies, which binds to drugs on RBC and induce complement mediated lysis of RBC and thus produce progressive anaemia. This drug induced haemolytic anaemia is an example of Type II hypersensitivity reaction.

Type III Hypersensitivity: Immune complex mediated hypersensitivity

When a huge amount of antigen enters into the body, the body produces higher concentrations of antibodies. These antigens and antibodies combine together to form insoluble complex called immune complex. These complexes are not completely removed by macrophages.

These get attached to minute capillaries of tissues and organs such as kidneys, lung and skin (Figure 11.9). These antigen-antibody complexes activate the classical complement pathway leading to vasodilation. The complement proteins and antigen-antibody complexes attract leucocytes to the area. The leukocytes discharge their killing agents and promote massive inflammation. This can lead to tissue death and haemorrhage.

Arthus reaction

It was first observed by Arthus. It is a local immune complex reaction occurring in the skin. Horse serum and egg albumin are the antigens that induce the arthus reaction. It is characterized by erythema, induration, oedema, haemorrhage and necrosis. This reaction occurs when antibody is found in excess. It appears in 2-8 hours after injection and persists for about 12-24 hours (Table 11.1).

Table 11.1: Difference between Immediate Hypersensitivity and Delayed Hypersensitivity

It is often called as delayed hypersensitivity reaction as the reaction takes two to three days to develop. Type IV hypersensitivity is involved in the pathogenesis of many autoimmune and infectious diseases such as tuberculosis and leprosy. T lymphocytes, monocytes and macrophages are involved in the reaction. Cytotoxic T Cells cause direct damage whereas the T helper cells secrete cytokines and activate monocytes and macrophages and cause the bulk damage (Figure 11.10).

Type IV hypersensitivity: Cell Mediated Delayed Hypersensitivity

Tuberculin reaction (Mantoux Reaction)

When a small dose of tuberculin is injected intra dermally in an individual already having tubercle bacilli, the reaction occurs. It is due to the interaction of sensitized T cell and tubercle bacterium. The reaction is manifested on the skin very late only after 48-72 hours.

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Immunology of Western Blot Techniques

Macromolecules immobilized or fixed on nitrocellulose membrane i.e., blotted can be subjected to a variety of analytical techniques more easily. Southern blotting was the first blotting technique developed which made the analysis and recording of DNA easy.

Later the technique was extended for analysis of RNA and proteins and they have acquired the jargon terms Northern and Western Blotting respectively.

Western blotting is also known as immunoblotting because it uses antibodies to detect the protein. Western blotting is a quantitative test to determine the amount of protein in sample.

Principle

Western blotting technique is used for the identification of a particular protein from the mixture of a proteins. In this method, the proteins are first extracted from the sample. Extracted proteins are subjected to Poly Acryl – amide Gel Electrophoresis (PAGE).

Transfer of proteins from poly acryl amide to the nitrocellulose paper is achieved by applying electric field. When radio labelled specific antibody is added on such membrane it binds to the specific complementary protein. Finally the proteins on the membrane can be detected by staining or through ELISA technique.

Steps

Step I:
Extraction of Protein

The most common protein sample used for Western blotting is cell lysate. The protein from the cell is generally extracted by mechanical means or by adding chemicals which can lyse the cell. The extraction step is termed as tissue preparation.

Protease inhibitor is used to prevent the denaturing of proteins. Using spectroscopy the concentration of the protein sample is analysed and diluted in loading buffer containing glycerol. This will help the sample to sink in the well. Bromothymol blue is used as tracking dye and is used to monitor the movement of the sample.

Step II:
Gel electrophoresis

The protein sample is loaded in well of SDS-PAGE (Sodium dodecyl sulfatepoly-acryl amide gel electrophoresis). The proteins are separated on the basis of electric charge, isoelectric point, molecular weight, or combination of all these. Proteins are negatively charged, so they move toward positive (anode) pole as electric current is applied. Smaller proteins move faster than the larger proteins.

Step III:
Blotting

Blotting refers to the transfer of the protein from the gel to the nitrocellulose paper by capillary action. Electro blotting is done nowadays to speed up the process. In electro-blotting nitrocellulose membrane is sandwich between gel and cassette of filter paper and then electric current is passed through the gel causing transfer of protein to the membrane.

Step IV:
Blocking

The nitrocellulose membrane is nonspecifically saturated or masked by using casein or Bovine serum albumin (BSA) before adding the primary antibody. This blocking step is very important in western blotting as antibodies are also proteins and they are likely to bind to the nitrocellulose paper.

Step V:
Treatment with primary and secondary antibody

The primary antibody is specific to desired protein so it forms Ag-Ab complex. The secondary antibody is enzyme labelled and is against primary antibody (antiantibody) so it can bind with Ag-Ab complex. Alkaline phosphatase or Horseradish peroxidase (HRP) is labelled
with secondary antibody.

Step VI:
Treatment with suitable substrate

Finally, the reaction mixture is incubated with specific substrate. The enzyme convert the substrate to give visible coloured product, so band of colour can be visualized in the membrane (Figure 11.6).

Application

1. The size and concentration of protein in given sample is determined by western blotting.
2. It is used in the detection of antibody against virus or bacteria in serum and helps in the disease diagnosis.
3. Western blotting technique is the confirmatory test for HIV. It detects anti HIV antibody in patient’s serum.
4. Useful to detect defective proteins.

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Antigen Antibody Reactions and its types | Working principle, Applications

The interaction between antigen and antibody is called antigen-antibody reactions. It is abbreviated as Ag-Ab reaction. This reaction is the basis of humoral immunity. The antigen and the antibody react to form immune complex.

Ag + Ab …………….. Ag – Ab complex The reaction between antigen and antibody is highly specific. It is compared to the lock and key system. The part of the antigen that combines with the antibody is called epitope or antigenic determinant. The part of antibody which combines with the antigen is called paratope or antigen determining site. Most of the antibodies have two binding sites and IgM has 5-10 binding sites.

Immunofluorescence

When antibodies are mixed with the fluorescent dyes such as fluorescein or rhodamine, they emit radiation. This phenomenon of emitting radiation by antibodies labelled with fluorescent dye is called immuno fluorescence. This reaction is well observed under fluorescent microscope. It is used to locate and identify antigens in tissues.

Types of Immunofluorescence

• Direct method
• Indirect method

Direct Method

In this method, the antibody labelled with fluorescent dye is directly applied on the tissue section. The labelled antibody binds with specific antigen. This can be observed under the fluorescent microscope.

Indirect Method

In this method, unlabelled antibodies are directly applied on the tissue sectionswhich bind with the specific antigens. Then the antibody labelled with the fluorescent dye is added to the tissue. Anti-antibody specifically binds with already added or linked unlabelled antibody (Figure 11.1).

ELISA (Enzyme Linked Immuno Sorbent Assay)

ELISA (Enzyme-Linked Immuno Sorbent Assay) is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. It is also known as Enzyme Immuno Assay (EIA).

In 1971, after the descriptions of Peter Perlmann and Eva Engvall at Stockholm University in Sweden, ELISA has become the system of choice when assaying soluble antigens and antibodies. All assays for antibody production depend upon the measurement of interaction of elicited antibody with antigen.

Principle

The principle of ELISA is very simple. The test is generally conducted in micro titre plates. (Figure 11.2 Micro titre plate).

If the antigen is to be detected the antibody is fixed in the micro titre plate and vice versa. Test sample is added in the microtitre plate, if there is presence of Ag or Ab in the test sample, there will be Ag-Ab reactions (with immobilized Ab or Ag). Later enzyme labelled antibody is added in the reaction mixture, which will combine with either test antigen or Fc portion of test antibody.

The enzyme system consists of:

1. An enzyme:

Horse Radish Peroxidase(HRP), alkaline phosphatase which is labelled or linked, to a specific antibody.

2. A specific substrate:

• O-Phenyl-diaminedihydrochloride for peroxidase
• P nitrophenyl Phosphate – for Alkaline Phosphatase

Substrate is added after the antigenantibody reaction. The enzyme hydrolyses the substrate to give a yellow colour compound in case of alkaline phosphatase (Figure 11.3). The intensity of the colour is proportional to the amount of antibody or antigen present in the test sample, which can be quantified using ELISA reader
(Figure 11.4 ELISA reader)

Types

There are four kinds of ELISA assay tests. They are: Direct ELISA, Indirect ELISA, Sandwich ELISA and Competitive ELISA (Figure 11.5).

i. Direct ELISA

An antigen is immobilized in the well of an ELISA plate. The antigen is then detected by an antibody directly conjugated to an enzyme such as HRP. Direct ELISA detection is much faster than other ELISA techniques as fewer steps are required.

The assay is also less prone to error since fewer reagents and steps are needed, i.e. no potentially cross-reacting secondary antibody needed. Finally, the direct ELISA technique is typically used when the immune response to an antigen needs to be analyzed.

ii. Indirect ELISA

Indirect ELISA is used to detect antibody. A known antigen is coated on the micro titre plate. If the patient’s serum contains antibody specific to the antigen, the antibody will bind to the antigen.

After incubation the wells are washed and the enzyme labelled anti Human Gamma Globulin (HGG) is added to the well. AntiHGG can react with antigen antibody complex. The substrate for the enzyme is added finally which is hydrolysed by the enzyme which develops a colour.

iii. Sandwich ELISA

Sandwich ELISA is used to detect antigen. A known antibody is coated on the micro titre plate. A test antigen is added to each well and allowed to react with the bound antibody. If the patient’s serum contains antigen specific to the antibody, the antigen will bind to the antibody.

Specifically bound antigen and antibody will remain in the wells even after washing. The second antibody is added and allowed to react with bound antigen. Substrate is added to measure colour reaction.

iv. Competitive ELISA

It is used for the detection of antigens. Antibody is first incubated with a sample-containing antigen. The antigen and antibody complex is added to the antigen coated microtitre well. If more antigen present in the sample, the less free antibody will be available to bind to the antigen coated well.

Addition of an enzyme conjugated secondary antibody specific to the primary antibody can be used to determine the amount of primary antibody bound to the well. It is a quantitative test for the antigen detection.

Application

An ELISA test may be used to diagnose:

HIV, Lyme disease, pernicious anaemia, Rocky Mountain spotted fever, rotavirus, squamous cell carcinoma, syphilis, toxoplasmosis, varicella-zoster virus, which causes chickenpox and Zika virus.

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Overview of Arbo Virus and its Various Types

Arbo Viruses (arthropod – borne viruses) are viruses of vertebrates biologically transmitted by hematophagous insect vectors. They multiply in blood sucking insects and are transmitted by bite to vertebrate hosts. Arbo viruses are worldwide in distribution.

Arbo viruses have been named according to the disease caused (yellow fever), the place of isolation of the virus (kyasanur forest disease) or the local name for the disease (chikungunya). They are classified into Toga, Flavi, Bunya, Reo and Rhabdovirus families.

Arbo viruses have a very wide host range including many species of animals and birds. The most important arbo virus vectors are mosquitoes, followed by ticks. The virus enters the body through the bite of the insect vector.

After multiplication in the reticuloendothelial system, viremia of varying duration occurs, or the virus is transported to the target organs such as central nervous system in encephalitis, the liver in yellow fever and the capillary endothelium in hermorrhagic fever.

Clinical syndromes are fever with or without rash, encephalitis, hemorrhagic fever, systemic disease and yellow fever. Diagnosis may be established by virus isolation or serology.

Samples (Blood, CSF) are inoculated intra cerebrally into sucking mice. The animal develop fatal encephalitis. Viruses may be isolated in tissue cultures or in eggs. Isolates are identified by hemagglutination inhibition, complement fixation, gel precipitation, immunofluorescence and ELISA. Virus isolated from insect vectors and from reservoir animal.

Toga Viruses

Toga viruses are spherical enveloped viruses with a diameter of 50-70nm. Single stranded RNA genome. The virus replicates in the cyloplasm of the host cell and released by budding through host cell membranes. The name Toga Virus is derived from ‘toga’ meaning the Roman Mantle refers to the viral envelope.

The genus Alpha Virus was formerly classified as Group A arbo viruses which explains the name Alpha Virus. The genus Alpha Virus contains 32 species of which 13 infect humans. All are mosquito borne.

Chikungunya Virus

The virus was first isolated from human patients of Aedes aegypti mosquitoes (Figure 10.9) from Tanzania in 1952. The name Chikungunya is derived from the native word for the disease in which the patient lies ‘doubled up due to severe joint pains’. The virus first appeared in India in 1963 in Calcutta, Madras and
Other areas.

The disease presents as a sudden onset of fever, Crippling joint pains, lymphadenopathy and conjunctivitis. A maculopapular rash in common. The fever is typically biphasic with a period of remission after 1-6 days of fever. The vector is Aedes aegypti. No animal reservoir has been identified. Antibody to the virus has been demonstrated in horses, cattle and other domestic animals.

Flavi viruses

The family flaviviridae contains only one genus flavivirus. They are smaller than alpha viruses, being 40nm in diameter. There are over 60 arthropod borne flava viruses classified as mosquito-borne and tick borne viruses. Examples of mosquito borne group known as encephalitis viruses they are St.

Louis encephalitis Virus, Ilheus virus, west nile virus, murray valley encephalitis virus and Japanese encephalitis. Tick borne viruses are classified in to tick borne encephalitis viruses and tick borne hemorrhagic fevers.

Dengue

The name dengue is derived from the ‘Swahili ki denga pepo’, meaning a sudden seizure by a demon. Dengue fever is similar to the illness caused by chikungunya. Four types of dengue virus exist: DEN1, DEN2, DEN3 and DEN4.

Dengue presents after an incubation period of 3-14 days as fever of sudden onset with headache, retrobulbarpain, conjunctival injection, pain in the back and limbs (break bone fever), lymphadenopathy and maculopapular rash. The fever is typically biphasic (saddle back) and lasts for 5-7 days.

Dengue may be more serious forms with hemorrhagic manifestations (dengue Hemorrhagic fever) or with shock (dengue shock syndrome). Dengue virus is transmitted from person to person by Aedes aegypti mosquitoes. The Incubation period is 8-10days. All four types of dengue virus are identified. Demonstration of circulating IgM antibody provides early diagnosis. IgM ELISA test offers reliable diagnosis. Difference between Dengue and Chikungunya is given in Table 10.2.

Difference between Dengue and Chikungunya

Zika Virus

Zika virus is a mosquito-borne flavivirus that was identified in Uganda in 1947 in monkeys. Zika spreads by daytime-active Aedes mosquitoes, such as A. aegypti and A. albopictus. The infection is known as Zika fever or Zika virus disease. Zika is related to the dengue, yellow fever, Japanese encephalitis, and West Nile viruses.

Zika virus is enveloped and icosahedral and has a non segmented, single-stranded, positive-sense (+) RNA genome (Figure 10.10). A positive-sense RNA genome can be directly translated into viral proteins, the RNA genome encodes seven nonstructural proteins and three structural proteins. One of the structural proteins forms the envelope. The RNA genome forms a nucleocapsid along with copies of the 12-kDa capsid protein.

Viral genome replication depends on the making of double-stranded RNA from the single-stranded, positive-sense RNA (ssRNA(+)) genome followed by transcription and replication to provide viral mRNAs and new ssRNA(+) genomes.

Pathogenesis and Clinical features

Zika virus replicates in the mosquito’s mid gut epithelial cells and then its salivary gland cells. After 5-10 days, the virus can be found in the mosquito’s saliva. If the mosquito’s saliva is inoculated into human skin, the virus can infect epidermal keratinocytes, skin fibroblasts in the skin and the Langerhans cells. The pathogenesis of the virus is hypothesized to continue with a spread to lymph nodes and the bloodstream.

Zika virus is primarily transmitted by the bite of an infected mosquito from the Aedes genus, mainly Aedes aegypti. The mosquitoes usually bite during the day, peaking during early morning and late afternoon or evening. This is the same mosquito that transmits dengue, chikungunya and yellow fever.

Zika virus is also transmitted from mother to fetus during pregnancy, through sexual contact, transfusion of blood and blood products, and organ transplantation.

The incubation period of Zika virus disease is estimated to be 3-14 days. The majority of people infected with Zika virus do not develop symptoms. Symptoms are generally mild including fever, rash, conjunctivitis, muscle and joint pain, malaise, and headache, and usually last for 2-7 days.

Zika fever (also known as Zika virus disease) is an illness caused by the Zika virus. Zika virus infection during pregnancy is a cause of microcephaly and other congenital abnormalities in the developing fetus and newborn. Zika infection in pregnancy also results in pregnancy complications such as fetal loss, stillbirth, and preterm birth.

Laboratory diagnosis

Virus can be demonstrated from the blood or other body fluids, such as urine or semen. Zika virus grow well in a variety of mammalian and insect cell lines. Zika virus is identified by NAAT – Nucleic acid Amplification test, Zika Antigen is detected by ELISA and PCR. Zika Antibody IgM is detected by MAC ELISA, IgG by ELISA and by PRNT plaque reduction neutralization test.

Prevention and Treatment

Protection against mosquito bites during the day and early evening is a key measure to prevent Zika virus infection. It is important to eliminate these mosquito breeding sites, Health authorities may also advise use of larvicides and insecticides to reduce mosquito populations and disease spread.

There is no treatment available for Zika virus infection or its associated diseases. No vaccine is yet available for the prevention or treatment of Zika virus infection. Development of a Zika vaccine remains an active area of research.

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Human Immuno Deficiency Virus

Human Immuno Deficiency Virus (HIV), the etiological agent of AIDS, belongs to the lentivirus subgroup of the family Retroviridae.

Structure

HIV is a spherical enveloped Virus, about 90-120 nm in size. The nucleo capsid has an outer icosahedral shell and an inner cone shaped core, enclosing the ribonucleo proteins. The genome is diploid, composed of two identical single stranded, positive sense RNA copies.

When the virus infects a cell, the Viral RNA is transcribed by the reverse transcriptase enzyme, first into single stranded DNA and then to double stranded DNA (provirus) which is integrated into the host cell chromosome. The virus coded envelope proteins are the projecting knob like spikes which binds to the CD4 receptors on susceptible host cells (Figure 10.8)

Viral Genes and Antigens

The genome of HIV contains the three structural genes (gag, pol and env) as well as other nonstructural and regulatory genes specific for the virus. These products of these genes, both structural and non structural act as antigens.

Genes coding for structural proteins

1. The gag gene → Determines the core and shell of the Virus. Precursor protein, p⁵⁵ and it is cleaved into three proteins p¹⁵, p¹⁸ and p²⁴. Major core antigen p²⁴ can be detected in serum.

2. The env gene → Determines the synthesis of envelope glycoprotein gp¹⁶⁰. Cleaved in to gp¹²⁰ and gp⁴¹

3. The pol gene → Codes for the reverse transcriptase and other viral enzymes such as protease and endonucleases. It’s expressed as a precursor protein, which is cleaved into protein p31, p51 and p66.

Pathogenesis

Infection is transmitted when the Virus enters the blood or tissues of a person and comes into contact with a suitable host cell, principally the CD4 lymphocyte. The receptor for the virus is the CD4 antigen and therefore the virus may infect any cell bearing the CD4 antigen on the surface.

Specific binding of the virus to CD4 receptor is by the envelope glycoprotein gp¹²⁰. Cell fusion is brought about by transmembrane gp⁴¹. After fusion with the host cell membrane, the HIV genome is uncoated and internalized into the cell.

Viral reverse transcriptase mediate transcription of its RNA into double stranded DNA, which is integrated into the genome of the infected cell through the action of the viral enzyme integrase, causing a latent infection.

The primary pathogenic mechanism in HIV infection is the damage caused to the CD4+T lymphocyte. The T₄ cells decrease is numbers. Infected T₄ cells do not release normal amounts of interleukin, gamma interferon and other lymphokines, this is damping effect on cell mediated immune response.

Clinical Features

AIDS is only the last stage in the wide spectrum in HIV infection.

1. Acute HIV infection

3-6 weeks of infection, persons experience low grade fever, malaise, headache, lymphadenopathy, with rash. Antibodies are usually negative at the onset of the illness but become positive during its course called ‘Sero conversion illness’.

2. Asymptomatic or latent infection

All HIV infected persons, whether or not they experience Sero conversion illness, pass through a phase of symptomless infection which may last up to several years. The infection progresses in course of time through various stages, CD4 lymphocytopenia, minor opportunistic infections, AIDS-related complex (ARC),
ultimately terminating to AIDS.

3. Persistent generalized
lymphadenopathy (PGL)

It is defined as the presence of enlarged lymph nodes at least 1cm, in diameter in two or more non contiguous extrainguinal, sites that persists for at least three months.

4. AIDS related complex (ARC)

This group includes patients with considerable immunodeficiency, suffering from various symptoms or minor opportunistic infections. eg. Oral candidiasis, Salmonellosis or Tuberculosis.

5. AIDS

End-stage disease, poor immune defence mechanism leading to the opportunistic infection and malignancies.

a. Commonest symptoms

Drycough, dyspnea and fever. Pheumonia may be viral (cmv) or fungal (Cryptococcus, Histoplasma).

b. Gastrointestinal system

The mouth is often involved with thrush, stomatitis, gingivitis, hairy leukoplakia. Dysphagia due to esophageal Candidiasis. Intestinal pathogen in AIDS is cryptosporidium. Other pathogens are Salmonellae, Mycobacteria, CMV or adeno viruses. ‘Gay bowel syndrome’ is common among the male homosexuals.

c. Central nervous system

The typical CNS opportunistic infections are toxoplasmosis and cryptococcosis. Lymphomas of the CNS are Common.

d. Malignancies

Kaposi’s Sarcoma was the lesion seen in male homosexuals. The tumours commonly seen are lymphomas, both the Hodgkin and non Hodgkin types.

e. Cutaneous

Herpes lesions, Candidiasis, Dermatitis, impetigo are common cutaneous lesions.

6. Dementia

Direct cytopathogenic damage in the CNS. It cross the blood-brain barrier and cause encepthalopathy leading to dementia.

7. Pediatric AIDS

Viral transmission may occur to the fetus in pregnancy. Many of the infected children may not survive for a year. Children may also acquire the infection from blood transfusion or blood products.

Laboratory Diagnosis

Lab diagnosis of HIV infection include tests for immuno deficiency in HIV infection.

A. Immunological tests

• Total leukocyte and lymphocyte count to demonstrate leucopenia and a lymphocyte count usually below 2000/mm³.
• Platelet count will show thrombocytopenia.
• Raised IgG and IgA levels

B. Specific tests for HIV infection

1. Antigen detection

Single massive infection, as by blood transfusion, the virus antigens may be detectable in blood after about two weeks. The major core antigen p24 is the virus marker in blood.

2. Polymerase Chain reaction

It is the most senstitive and specific test.

3. Antibody detection

Demonstration of antibodies is the simplest and widely employed technique. It takes 2-8 weeks to months for antibodies to appear after infection, during this period, the individual may be highly infectious. This sero negative infective stage is known as the ‘window period’. Antibody can be detected by

1. ELISA
2. Western blot test.

Treatment

The treatment of AIDS include:

• The treatment and prophylaxis of infections and tumours
• General management
• Immunorestrorative measures
• Specific anti-HIV agents

Effective drugs are available, they are Zidovudine, Didanosine, Zalcitabine, Lamivudine and Protease inhibitors like Saquinavir, Ritonavir, Indinavir used as monotherapy or in various combination.

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Rabies Virus Types and its Laboratory Diagnosis

The Family Rhabdoviridae contains viruses that infects mammals, reptiles, birds, fishes, insects and plants. The disease in human being is called hydrophobia because the patient exhibits fear of water, being incapable of drinking though subject to intolerable thirst.

Pasteur established that the rabies virus was present in the brain of infected animals. By serial intracerebral passage in rabbits, he demonstrated fixed virus that could be rendered immune by a series of injections. Vaccine was prepared by drying pieces of spinal card from rabbits infected with the fixed virus.

Joseph Meister a nine year old boy, severely bitten by a rabid dog and in grave risk of developing rabies, was given a course of 13 inoculations of the infected cord vaccine by Pasteur. The boy survived. This dramatic event was a mile stone in the development of medicine.

Morphology

The rabies virus is bullet shaped, with one end rounded or conical and the other planar or concave. The lipoprotein envelope, carries knob like spikes, composed of glycoprotein G responsible for pathogenesis, virulence and immunity beneath the envelope is the matrix (M) protein layer which may be invaginated at the planar end. The membrane may project outwards forming a bleb. The genome is unsegmented linear RNA (Figure 10.6).

The rabies virus isolated from natural human or animal infection is termed ‘the street virus’. Rabies has been recognized from very ancient times as a disease transmitted to humans and animals by the bite of ‘mad dogs’. The name rabies comes from the Latin word rabidus, meaning ‘mad’, derived from the Sanskrit root rabhas, for frenzy.

Pathogenesis

Human infection is usually caused by the bite of rabid dogs or other animals. The virus present in the saliva of the animal is deposited in the wound (Figure 10.7). Rarely, infection can also occur following non-bite exposures such as licks or aerosols.

The virus appears to multiply in the muscles, connective tissue or nerves at the site of deposition for 48-72 hours. It penetrates the nerve endings and travels in the axoplasm towards the spinal cord and brain, at speed of about 3 mm per hour. The virus multiples and spreads centrifugally along the nerve trunks to
various parts of the body including the salivary glands. It multiplies in the salivary glands and is shed in the saliva.

The virus reaches every tissue in the body and dissemination may be interrupted at any stage by death. In humans the incubation period is usually from 1-3 months, short as 7 days or as long as three years. The incubation period is usually short in persons bitten on the face or head and long in those bitten on the legs. This may be related to the distance the virus has to travel to reach the brain. The incubation period is generally shorter in children than in adults.

The four stages of the disease are as follows, prodrome, acute encephalitic phase, coma and death. The onset is marked by symptoms such as fever, headache, malaise, fatigue and anorexia, anxiety, agitation, irritability, nervousness, insomnia or depression. The neurological phase begins with hyperactivity. Attempts to drink during such painful spasms of the pharynx and larynx produce choking or gagging that patients develop a dread of even the sight or sound of water (hydrophobia).

Animal Infection

In dogs, the incubation period is usually 3-6 weeks but it may range from 10 days to a year. The initial signs are an alert,troubled air and restlessness,snapping at imaginary objects, licking or gnawing at the site of the bite. After 2-3 days of this prodromal stage, the disease develops into either the furious or dumb types of rabies. In furious rabies, dog runs biting without provocation, the lower jaw droops and saliva drools from the mouth.

Paralysis convulsions and death follow. In dumb rabies, is the paralytic form, animals lies huddled, unable to feed. About 60% of rabid dogs shed the virus in saliva.
Rabid dogs usually die in 3-5days.

Laboratory Diagnosis

Human Rabies

The specimens tested are corneal smears and skin biopsy. Commonly used method for diagnosis is the demonstration of rabies virus antigens by immuno fluorescence. Direct immunofluorescence is done using antirabies serum tagged with fluorescein isothiocyanate.

Negri bodies in the brain, are demonstrated, Isolation of the virus by intracerebral inoculation in mice can be attempted from the brain, CSF, saliva and urine. The mice are examined for signs of illness, and their brains are examined after death.

Animal Rabies

The whole carcassof the animal suspended to have died of rabies may be sent to the laboratory. The brain may be removed sent for biological test and microscopy respectively. The portion of brain sent should include the hippocampus and cerebellum as negri bodies are most abundant. The following tests are done in the laboratory.

1. Demonstration of rabies virus antigen by immuno fluorescence
2. Demonstration of inclusion bodies – Negri bodies are seen as intracytoplasmic, round or oval purplish pink with characteristic basophilic inner granules. Negribodies vary in size from 3.27 Mm.

Antirabic Vaccines

Antirabic vaccines fall into two main categories neural and non-neural.

Neural Vaccines

Suspension’s of nervous tissues of animals infected with the fixed rabies virus. Following are the modified forms.

1. Semple Vaccine:

Vaccine developed by semple (1911). It is a 5% suspension of sheep brain infected with fixed virus and inactivated with phenol at 37°C leaving noresidual live virus.

2. Beta propiolactone (BPL) Vaccine:

Beta propiolactone is used as the inactivating agent instead of Phenol.

3. Infant Brain Vaccine:

The enceptalitogenic factor in brain tissue is a basic protein associated with myelin. Vaccines were developed using infant mouse, rat or rabbit brain. Infant brain vaccine is impractical in India.

Non-neural Vaccines

Non-neural vaccines includes

1. Egg Vaccines
2. Tissue Culture Vaccines
3. Subunit Vaccine

Passive Immunisation

Human rabies immune globulin (HRIG) is free from the danger of sensitization but should be ensured free from HIV and hepatitis viruses.

Vaccines for Animals

Antirabies immunization in animals is to be done as pre-exposure prophylaxis concentrated cell culture vaccines – inactivated virus gives good protection after a single Intramuscular injection. Injections are given at 12 weeks of age and repeated at 1-3 years intervals.