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The features of the disease provided such as presence of flu like symptoms followed by anemia, lethargy ,high fever , weight loss infection, impaired immune response , elevated white cell count, metastatic lymphoma and prominent occurrence in bi- sexual and heterosexuals in compare to that of homosexuals. These symptoms suggest at a preliminary level that causative agent is a virus and not any other agent such as parasite etc. the confirmation of which can only be done after diagnostic tests. Viruses cause disease by 2 different ways :
Depending on the type of viral products made, host factor induced and type of replication. They may disturb the internal membranes causing the lysis of cell to to release of autolytic enzymes. This will lead to imbalanced passage of fluids and ions causing sell death or distortion.
Viruses can also affect the function of cell which is a less significant effect which causes disease and not cell death. ( REF AVP) Here the viruses were found to invade only differentiated cells such as lymphocytes, neurons and endocrine cells etc. ollowed by altering the synthesis of immunoglobins, neurotransmitters, harmaones and lymphokines. Here the cell function appears to be normal under microscopy techniques as well as cell growth assays. But eventually presence of disease is detected as less quatity of the differentiated product is produced such as growth harmone, immunoglobin, insulin etc. which leads to imbalancing of homeostasis and occurrence of disease such as growth retardation, virus persistence and diabetes.
Table 1: Replication frequency determines whether that infection will spread thus becoming a systemic infection or remain localized in virus at site of entry.
Lymphoid tissues, C.N.S.
Oropharynx or G.U.tract
Lymphoid cells, C.N.S.
Some of the features of the disease give a clue about the possible mechanism of the disease which may be similar to that of Human Deficiency Syndrome. It is now known that the two types of HIV that is HIV-1 and HIV-2 are mainly transmitted from mother to infant and by heterosexual intercourse (Rambaut et al., 2004). The multifactorial and complex mechanism of this disease has been extensively studied. It was shown that due to secretion of many cytokines the virus expression is upregulated and the induction of intensive cell signalling by viral envelope takes place. The primary receptor is CD4 molecule for the virus on subset of monocytes and T cells. Lymphoid tissue is the main target and acts as reservoir for HIV.
Figure 1: HIV (pink) when enters the body it binds to dendritic or Langerhans cells (orange). These cells carry the HIV particle to CD4+ cells. The infected CD4+ cells reside in lymphoid tissue where mainly the infection spreads. With the acceleration of virus replication throughout the lymphoid tissue dissemination of virus takes place. HIV specific immune response takes place and virus is placed on follicular dendritic cells. At this point the persistent chronic infection is established. Eventually lymphocyte depletion takes place and architecture of the lymphoid tissue is destroyed. (Fauci, 2003).
The featured symptom elevated white cell count and highly metastatic lymphoma can be associated with a well studied example of "Molecular Mechanisms of Human T-cell Leukemia/Lymphotropic Virus, Type I Infection By Genoveffa Franchini." HTLV 1 (Human T-Cell Leukemia).This virus was discovered before the discovery of HIV. There is a long latency period before the induction of T-cell Leukemia. The occurrence of malignancies is generally found to take place in ages of 18 - 28. http://bloodjournal.hematologylibrary.org/cgi/reprint/86/10/3619.pdf
The flu like symptoms are strongly suggestive of (SLE) systemic lupus erythematosus. In a case described by Cope et al, 1992 a 59 year old female patient was suffering from Human Parnovirus (B19) which coincided with the onset of systemic lupus erythematosus. http://ard.bmj.com/content/51/6/803.full.pdf
Our understanding of virus caused infection has elevated dramatically over the past 2 decades. Analysis of RNA and DNA that has been extracted after autopsy and biopsy specimens by northern blotting and southern blotting techniques is helpful is confirming the presence of virus. These techniques when used in combination with PCR (polymerase chain reaction) proved to be highly sensitive so that they were even able to detect the low copy number of virus in sample. Although PCR is very efficient identification of the infected cells is still a limitation of this technique. In contrast to this, immunocytochemistry and in situ hybdridization are exquisitely techniques to localize virus gene product and viruses in cells. Here, we discuss the application of polymerase chain reaction (PCR) , immunocytochemistry and in situ hybridization in detection of disease.
There are mainly 3 major reasons for diagnosis of viral infection : ( MEDICAL VIROLOGY REF)
Where cure or treatment depend on diagnosis.
Where confirmation of reason aids in handling the patient better by preventing use of strong unwanted medicines like antibiotics and sometimes ealrly discharge from medical centre or hospital.
For acquiring the epidemiological data.
In many case an efficient diagnosis can be can be made within 1 day unlike the case of cell cultures which takes weeks to grow and show identifiable infect ion. Conventionally many techniques were used for confirmation of viral infection such as electron microscopy, immunofluorescence etc.
For the preparation of specimen the slides must be acid cleaned followed by Poly D lysine coating. This coating allows the cells or sections to readily attach to surface of slide. If the slides are not pre-coated this may lead to loss of specimen.
When the cells or section have been attached and fixed to slide ( coated by poly D lysine), immunochemical techniques are applied. Here we will detect the immunoreactive sites present on cells and tissues using special antibodies which are directed against polypeptides or proteins of virus. We can use polyclonal as well as monoclonal antibodies. This techniques has 2 detection systems. In the Immunoperoxidase detection system following scheme will be used:
Application of antibody ( conjugated to biotin) specific for a specific antigen determinant has to be applied to slide.
In case the primary antibody is not found to be biotinylated then another biotetinylated antibody ( secondary antibody) is applied.
The biotin-avidin complex is now applied to the slide.
The reaction of chromagen with biotin-avidin takes palce. This reaction is catalysed by peroxidase H2o2 to produce a stain. Here the commonly used chromagens are 3 amino- 9 ethylycarbazole (AEC) and 3,3' diaminobenzadine (DAB).
The technique of in - situ hybridization is used to screen cells and tissues for detection of presence of viral DNA or RNA. The viral nucleic acid specific fragments which are labelled with 35S so that it can be used as radioactive probe to detect the presence of viral nucleic acids. Some other types of isotopes such as 3H and 32P can also be used but best results are given by 35S. Whole in situ process can be wrapped in 6 days. The first day is marked by the pre treatment of slides to allow entry of radioactive probe into cell or tissue. This is preceded by overnight hybridization of slide with probe. On the following day washes are performed to remove non specific bound probes. On the very same day the slides are applied with photographic emulsion which will develop over a period of 4 days. Now on the sixth day using fixer and standard developer the slides are developed. Ideally the slides should develop in 4 days only because if they develop in 3 days it is interpreted as there is a considerable reduction in signal on the other hand if 5 to 6 days mean thathigh amount of non-specific background.
A technique called as" immunochemistry - in situ double labeling" helps in detection of presence of both nucleic acids and proteins. This technique is very efficient in detecting specific types of cells that a affected as a result of pathogenesis. Here Immunochemistry is used as a tool to stain marker on cell surface whereas the in situ hybridization can be used to detect infection causing agent.
For the advanced diagnostic techniques such as PCR we will need to extract the DNA from the infected tissue. The procedure may involve the following steps: (AVP)
Resuspension of 200mg of tissue in digestion buffer (1 ml) followed by vigorous vortex.
Addition of proteinase K the final concentration ( 500µg/ml) now incubation will be performed after gentle mixing. Incubation temperature will be about 37-55oC. Generally in practice the sample is incubated at 480C for about 24 hours.
Now vortex is done followed by addition of SDS and proteinases K to final concentration of 1mg/ml and 2% respectively. Preceded by incubation for 7-10 days in case of DNA and additional 6-12 hours for RNA.
After completion of digestion DNA is extracted with help of 1:1 phenol/chloroform volumes and some modifications in salt concentration. Samples are kept at -200C overnight. The DMA is resuspended in TE buffer for further use.
The features of the disease seem to be highly specific thus we need to develop a highly efficient, specific and fast method of detection. We may use PCR techniques to detect the causative agent from patient specimen. There are some factors to be considered while preparation of primers. Such as high G/C content and absence of internal complimentarity. This diagnostic method may proceed somewhat like in case of detection of mycoplasma virus in respiratoty tract sample of a HIV positive patient by PCR ( Polymerase Chain Reaction. In this diagnostic technique the blood can be collected in a tube containing anticoagulant EDTA. Addition of about 1.25ml of blood in Ficol Histopaque solution will be done followed by centrifugation where separation of blood components depending upon differential density will take place.. Side by side it is ideal to make a positive ( for disease) control and a negative control (water). The PMBC fraction obtained after centrifugation with histopaque is used after processing in master mix in microlitre quanties. The master mix consists of deoxynucleosides triphosphates, DNA polymerase enzymes , primers that bind specifically to the presumed causative virus gene and a second set of primers which acts as internal control for specimens that will amplify a portion of the related human gene in all human cells such a Beta globin gene in case of detection of HIV http://www.uhl.uiowa.edu/publications/archive/hotline/1995/hivpcr.xml. The patients specimen should be positive for the PCR ( Polymerase Chain Reaction) product of the second set of primers so that the results can be interpreted correctly. Now the master mix plate is placed in thermo cycler to run 50 cycles which may last about some odd 3 hours after which specimens are subjected to agarose gel electrophoresis where the results are interpreted after staining with ethidium bromide and visualistaion under uv light. (http://jmm.sgmjournals.org/cgi/reprint/47/11/983.pdf)