Detection and identification of viral genetic sequences

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It was found that viral infections are a cause of 15 -20% of human cancers and in many cases viral genetic sequences were isolated from these sites of infection. JC Polyomavirus and SV40 are known to encode large T antigens and small t antigens. These antigens are viral oncogenes and are able to inhibit apoptosis, induce cell proliferation and disturb cell cycle. JCV has been detected in human malignant tissues such as lung cancer [Giulian et.al 2009], colorectal cancer tissues, gastric cancer, esophagal cancer. SV40 is a polyoma virus which has been detected in Brain tumour, Bone tumour, pituitary gland tumour [Barbanti-Brodano et.al. 2004] and lung cancer tissues [Giulian et.al 2009]. These high estimations of presence of viral genetic sequences in human cancers have led researchers to investigate the role of these sequences and the modifications which these viral genetic sequences can undergo to cause carcinoma. Current study is aimed to detect the presence of viruses in prostate cancer tissues. For this formalin fixed prostate hyperplasia tissues and formalin fixed prostate carcinoma tissue slides were obtained for immunohistochemical testing. Immunohistochemical testing can be done for detecting the presence of large T antigen produced by BKV, SV40, XMRV and JC polyomavirus. Plasmid DNA transformation , Extraction and Purification was then Amplification of DNA can be done for detecting the Presence of genetic sequences by using primers specific for the BKV, XMRV, JC polyomavirus and XMRV.

INTRODUCTION:

It was found that viral infections can induce murine leukaemia in mice. Retroviral infections were known to cause lymphproliferative disorders in cattle and cats. Except HCV and retrovirus, all other human oncogenic viruses are DNA virus. Direct cell transformations can be induced by oncogenic viruses by insertional mutations or by oncogenes in the viral genome. Whereas some viruses can contribute for cell proliferation by disturbing the normal cell processes like apoptosis and suppressing the host immune system.[Zur hausen et.al. 2001]

Polyomavirus are highly host specific and human is the only host which is recognized for BKV and JCV, they show different human transmission and tissue dissemination patterns. When a Polyomavirus infects a human, it starts normal viral replication by regulating the host and its genes to replicate the viral DNA, which then lyse the cell & infection is continued. The other mechanism can be observed in non permissive host cells which do not let viral replications to continue or even begin. These type of Polyomavirus can result in continuous expression of viral early genes, which may lead to host cell transformation. It has been observed that the expression of large T antigens is greater in tumour cells than that in normal cells [Imperial M.Z. 2001]. Among Polyomavirus, large T antigen has been found to have greater effect on oncogenicity of the virus. Human Polyomavirus BKV, JCV and SV40 are homologus in their genomic structure. JCV DNA sequence is 72% identical to BKV DNA sequence, also BKV & SV40 show 69% similarity, and JCV and SV40 show 68% identical sequences.

XMRV genome is transcribed into an unspliced single transcript that will be translated to a polyprotein which have core proteins encoded by gag region, protease, integrase and reverse transcriptase encoded by gag region on the genome. Unlike other retrovirus, XMRV does not encode accessory proteins or host derived oncogenes. This virus is 90% identical to murine leukemia virus. [Silverman et.al. 2010]. Studies with XMRV have shown that a 270bp sequence found in between 5’gag leader and gag CTG alternative start codon, which codes for a 90 amino acid glycol-gag protein is not seen in Murine leukemia virus. This alteration could be a reason for the viral pathogenicity in humans [Urisman et.al 2006]. XMRV induces oncogenicity by insertional mutation, where it activates the host cell protooncogenes, followed by integration of its genome into the host cells. It has been shown that Gamma retrovirus cause malignancies in feline, rodents and primates, but XMRV is considered to have probable association with Prostate cancer [Schalberg et.al 2009].

Presence of XMRV in human prostate cancer individuals showed a deficiency of RNase L. RNase L is a ribonucleotide breaks down single stranded RNA molecules and is a interferon mediated antiviral response[Kim et.al 2010]. Some studies have shown that proviral insertional mutation can be mechanism of infection for SMRV related Prostate cancer. The integrated for of XMRV, the XMRV provirus has 4 base pair direct repeated sequences which can affect site recognition and host cell rearrangement like insertion or deletions. This could be another possible mechanism of transformation of host cells. Studies done by Sakuma [Sakuma et.al 2010] and Metzger [2009] showed that XMRV cannot induce cell transformation directly and it does this by inducing host cell oncogenes.

Prostate and bladder are part of the urinary system, Prostate surrounds the urethra and is closely located to the bladder. Benign prostatic hyperplasia is common problem and mostly seen in men above 50. Its development is related to prostatic cell death inhibition which lead to continuos proliferation of cells. Inhibition is related to the androgen , dihydrotestosterone. Its severity ranges from carcinoma without clinical symptoms to fatal metastatic tumours. Exact mechanism of Prostate cancer development is not known but is considered to be a multistage process. The cancer develops from interepithelial neoplasia to prostate carcinoma and they show nuclear alterations loss of cellular components. Some molecular changes are observed in Prostatic interepithelial neoplasia and carcinoma cells[Kumar et al 2010].

MATERIALS AND METHODS:

Immunohistochemistry:

Formalin fixed paraffin embedded prostate hyperplasia tissues,formalin fixed paraffin embedded prostate carcinoma tissues can be subject to immunohistochemical staining protocols. This could be the best possible method to start with and its ease of use and detectable result can be useful in the study. Also the carcinogenesis pattern can be observed under the microscope and presence of large T antigen can be detected by using pab-416 antibody. For this protocol, the slides were first deparrafinized by immersing slides in 2 changes of xylene for 5 mins, and then rehydrating in absolute ethanol, 90% ethanol and 70% ethanol for 3 mins each. Sections then need to incubated in 3%H202 solution in methanol to inhibit endogenous peroxidise activity and then washed with water to remove any chemicals left. The slides then need to be immersed in PBS and incubated and boiled in preheated citrate buffer with EDTA and allowed to cool at room temperature. The sections then need to be blocked by 50% normal horse serum in PBS for 10 mins and then subject to primary antibody Sc-136172 for 60 mins. This antibody is specific to MCV large T or 57kt antigens. Similarly pab-416 antibody can be used for detection of SV40, BKV and JCV. Primary antibodies can then be detected by addition of secondary antibodies that may attach to specific antigens to form a Primary secondary antibody (Primary-secondary-Ab) complex. They can then be developed with DAB and following washing and differentiation with acid alcohol and dehydration with ethanol, slides can be mounted and observed under microscope for presence of formation of avidin â€" biotin complexes. Differences in normal tissues and Carcinoma can also be observed and the structural characteristics can be noted. This study can then help in the further studies for PCR amplification.

DNA transformation, Extraction and Purification:

For cells to take up external DNA , they first need to be made permeable so that they can take up external DNA. This state is generally referred to as competency and cells are known as competent cells. Competency can be induced by treatment with chloride salts, metal cations or cold treatment but these treatments can be lethal to the cells and care has to be taken not to damage the cells. E-coli can be transformed by ampicillin which damages the membranes by crosslinkage of membrane proteins. Cells exposed to ampicillin will grow only if the plasmid containing the gene of interest are carrying gene for ampicillin resistance. Plasmid DNA needs to be mixed with competent cells and then given a heat shock and incubated on ice for 4 mins. The Tubes containing Plasmid DNA â€" competent cell mixture then needs to be incubated and transferred to LB agar plates containing 100ug/ml ampicillin and checked for growth after 24 hours. Kits are available for transformation and this transformation can be helpful in obtaining transformed cells for further Extraction and Detection.

Transformed cells then need to selected and grown in broth cultures containing 100ug/ml of ampicillin. These samples then need to be transferred in centrifuge tubes for extraction, where they are centrifuged to obtain a pellet and treated with Plasmid Extraction kits. The procedure can be carried out as per the manufacturer’s protocol to obtain DNA pellets in the tubes. The pellets obtained were then dissolved in TE buffer and concentration can be measured by measuring the absorbance of light by spectrophotometer.

It is necessary to obtain the viral genetic sequence in order to carry out PCR amplification of DNA. Hence we need to grow the viruses in transformed cells so that we can select and isolate the transformed cells carrying viral genome of interest. These cells then need to be lysed to extract DNA present in them and this can be done by using Kits available in the market. DNA samples thus obtained from this method can then be used for amplification by nested PCR.

Polymerase chain reaction protocol:

It is necessary to carry out a nested PCR for detection of viral plasmid. The samples are subject to two primer sets to obtain an amplification of viral protein genes. Past studies have already been done to amplify DNA of interest. Primers designed and used in earlier studies [Giraud et al 2008], [Feng et al 2008], can be used for detecting sample DNA. Samples obtained positive for MCV, XMRV, JCV, BKV can be used in the first round of nested PCR.

Products obtained from the first round of nested PCR are then used as DNA template in second round of PCR reaction. PCR products can then be transferred and visualised on agarose gels containing Ethidium bromide and visualised under UV transilluminator.

It is recommended to use a nested PCR instead of one step PCR in viral genetic sequences to increase the specificity. Also it is necessary to carry out nested PCR for the product to undergo sequencing and to obtain a clean sequence.

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