PREVALENCE OF INTEGRATED HBV DNA AMONG BLOOD DONORS IN EGYPT

lood transfusion service (BTS) is a necessary and key part of the medical services framework. The main objective of the BTS is to ensure safety, adequacy, availability, and effectiveness of blood supply at all levels (Islam, 2009). Transfusion of blood and blood derivatives, as a specific methodology of patient administration spares a large number of lives worldwide every year and decreases HBV transmission. It is outstanding that blood transfusion is associated with a substantial number of complexities, some are just trifling and others are conceivably life, undermining, requesting for fastidious pre-transfusion testing and screening. Unscreened blood transfusion expose the patient to infect with many transfusions transmitted infections (TTI) like hepatitis virus (HBV, HCV), human immuneinsufficiency virus (HIV), syphilis, malaria, etc. Transfusion units have dependably been a noteworthy entry to monitor, screen, and control diseases transmitted by blood transfusion. (Khan et al., 2007).

/VACSERA lood transfusion service (BTS) is a necessary and key part of the medical services framework. The main objective of the BTS is to ensure safety, adequacy, availability, and effectiveness of blood supply at all levels (Islam, 2009). Transfusion of blood and blood derivatives, as a specific methodology of patient administration spares a large number of lives worldwide every year and decreases HBV transmission. It is outstanding that blood transfusion is associated with a substantial number of complexities, some are just trifling and others are conceivably life, undermining, requesting for fastidious pre-transfusion testing and screening. Unscreened blood transfusion expose the patient to infect with many transfusions transmitted infections (TTI) like hepatitis virus (HBV, HCV), human immuneinsufficiency virus (HIV), syphilis, malaria, etc. Transfusion units have dependably been a noteworthy entry to monitor, screen, and control diseases transmitted by blood transfusion. (Khan et al., 2007). Hepatitis B virus (HBV) infection is a critical worldwide health problem influencing two billion individuals worldwide and 400 million people suffer from chronic HBV infection (Dhawan et al., 2008). HBV transmitted sexually, perinatally, and percutaneously, manipulate 350 to 400 million people around the world. HBV disease caused every year for 1 million deaths worldwide from liver failure, cirrhosis, and hepatocellular carcinoma. (Lee, 1997;Hoofnagle et al., 2007).

HBV
is a member of Orthohepadnavirus genus of the Hepadnaviridae family, which is classified to Retroid viruses (Kann and Gerlich, 1998). HBV genome around 3.2 KB, its smaller, incompletely double-stranded DNA genome, HBV has four highly overlapping open reading frames (ORFs) (Nassal, 1999;Seeger and Mason, 2000). HBV has a higher rate of mutation than other DNA viruses, because of using RNA polymerase without proofreading function. Thus, generally it is assumed that reverse transcription step is a source for the majority of point mutations and deletions or insertions that can be observed in the HBV genome (Petzold et al., 1999).

B
HBV DNA integration has been detected in HBsAg-negative patients (Brechoht et al., 1987).It has been recorded that HBV DNA could integrate into the host hepatocytes as well as the spermatozoa. (Hino et al., 1991;Peng et al., 1988).
HBV DNA usually integrates in cellular genes involving cell growth or signaling control. When HBV DNA integrates into the host chromosome chromosomal stability is also decreased (e.g., large inverted deletions, duplications and chromosomal translocation) (Chamberlain, 2016).

MATERIAL AND METHODS
Ninety thousand and one five hundred volunteer blood donors were collected from blood donation campaigns for the company blood transfusion services (VACSERA) during 2014. Blood samples are usually collected in EDTA tubes.
Then, samples were centrifuged and the plasma was transferred to a screw-cap cryo tube within 1 h of collection.

Nucleic Acid Test (NAT)
For the qualitative detection of HBsAg, Chemiluminescent immunoassay (ChLIA) was used. NAT test was used as a confirmatory test to screen samples with negative ChLIA of HBsAg in collected plasma. Samples, only which diagnosed as positive NAT for HBV with no coinfection with other hepatitis viruses such as HCV and HDV or HIV, had been aliquot and stored at -20C until use.

Extraction of HBV DNA and PCR amplification
DNA was extracted from two plasma samples; Total DNA was extracted from 200 μL plasma by using QIAamp DNA Blood Mini Kit (Qiagen, Cat. No. 5114) (Goodarzi et al., 2007).
PCRs designed to amplify the complete surface antigen of HBV. A simple and efficient way for virus primer design was used based on the alignment of HBV genotype sequences published at gene banks database (Stuyver et al., 2000). Taq PCR Master Mix kit (Qiagen, Cat. No. 201443) was used to amplify HBsAg. Final volume of PCR was 100 μl, according to the instruction of the manufacturer. Thermal profile of PCR reaction consist of: preheating at 95C for 5 min, 35 cycles of 95C for 45 sec, 60C for 40 sec, and 72C for 60 sec; finally at 72C for 10 min as a final extension step. 2% agarose gel with ethidium bromide stain (0.5 µg/ml) was used to separate the PCR products. PCR products were purified by using gel extraction Kit. (Qiagen, Cat. No. 28704).

Cloning and sequencing
When direct sequencing for amplified fragments were failed according to the protocol provided by the manufacturer of DNA sequence kit. The PCR products ≈1200 bp were cloned into pPCR -Script TM Amp SK + vector (Stratagene, La Jolla, sCA, USA) according to manufacture protocol, sequencing had been carried out at Genetic Engineering Research De-partment (VACSERA). BigDye Terminator v3. 1 Kit (Life Technologies) was used as a ready reaction to perfume cycle sequencing then read and analyzed on a 310 DNA automated sequencer (Life Technologies Inc.). Primers Used to read the sequence were T3 (5' -AATTAACCCTCACTAAAGGG-3') and T7 (5' -GTAATACGAC-TCACTATAGGGC-3') in the tow direction forward and reverse sequences were assembly and analyzed by using the NCBI Blast tool and to identify viral genome sequences, and to locate the integration sites in the human genome. (Takahashi et al., 1998).

RESULTS AND DISCUSSION
Insurance blood transfusion service and safety of blood products is one of the major issues in the area of transfusion medicine. Blood screening is playing a major role to decrease the risk of transfusion of infected units. Firstly, testing of antigen/or antibody markers of blood borne disease was established. Wherefore, limitation of these serological techniques including the window period between infection and detection, and antigenic variability enhance implementation of the second testing for nucleic acid technique (NAT). (Hollinger and Liang, 2001).
From ninety thousand and five hundred volunteer blood donors only two samples had a negative result for HBsAg by using Chemiluminescent immunoassay and diagnosed positive HBV for NAT test with no co-infection with other hepatitis viruses such as HCV, HDV or HIV.
Perchance, chromosomal Human DNA was amplified by using primers designed for viral DNA. In this study, two human chromosomal DNA were amplified by using primers designed to amplify surface antigen of HBV. These two samples carried integrated virus DNA. Both of them had both "left" and "right" intersection of the integrated virus DNA. The structure of integrated virus genomes were determined by constructing the sequence of cloned PCR product that amplified with specific primer for HBV.
The expected PCR Product size is 1.3 kb, the subgenomic PCR designed to amplify the surface antigen of HBV gave rise to smaller amplicons ranging in size from 300 to ≈1200 bp. Only amplicon with size ≈1200 bp were successfully cloned into a pPCR-ScriptTM Amp SK + vector after direct sequence failed to give a clear and significant reading (Fig. 1) and the two cloned samples were sequenced (Table 1).
DNA sequences were processed in the pPCR SK(+) plasmid containing amplicons soon after sequences were checked against a gene bank. The identities of the sequenced samples were 98% with human chromosome 5. Integration point in the 1 st and 2 nd samples were located and determined in Table (1) . Integration sites of HBV DNA in human HCCs have been found on almost all chromosomes, a random distribution throughout the host DNA (Tokino et al., 1991) this confirmed by finding of Slagle et al. (1992) that HBV DNA insertions are found at random sites in human cellular DNA.
Albeit a few chromosomes are affected more often than others in the present study four sites were found to be identical as integrated HBV DNA into Human chromosome 5 and fifth site locate in the 1st sample only. The length of integrated fragment ranging from 10 to 19 nucleotides which supported by the finding of Nagaya et al. (2015) who found that the inserted fragments HBV DNA were less than 24 bp. Sites of chromosomal fragile are large regions of DNA, which replicate late during S phase of the cell cycle and are predisposed to deletions or breaks. So hot spots for foreign DNA integration multiply. Unlike retroviruses, in which viral DNA inserted into host DNA is a step of replication cycle, integration does not carry out during normal Hepadnaviruses replication. Integration of Hepadnaviruses DNA may take place as a result of an illegitimate recombination mechanism mediated by cellular enzymes. (Yang and Summers, 1999;Wang and Rogler, 1991).
In the current study, the integration sites mapped to show direction of integrated fragment into chromosomal DNA (Fig. 2). Three intersections were integrat- was ranging from 20 to 35 years (Kunio, 1989).
Early studies confirmed that integration of HBV occurs in patients with occult HBV infection and with HCC (Lai et al., 1990). In most of cases, HBV integration was common in tumorous than in nontumorous tissue. (Matsuzaki et al., 1997;Matsuzaki et al., 1999) A higher rate of chromosomal abnormalities is found in HBV-related to HCC than can be clarified alone by the accumulation of chromosomal mutations.
HBV is an oncogenic virus and its DNA can be integrating into the genome of hepatocytes. Integration of HBV DNA sequences is present in about 80% of human HBV related HCCs. Integration of HBV DNA may confer an eclectic growth advantage on infected hepatocytes and leads to the onset of tumor progression. (Chamberlain, 2016).

SUMMARY
This study presented important data about the gap between serology screening (surface antigen) and nucleic acid testing (NAT) testing of blood donors. Effectiveness of NAT for blood donors screening is a debating area in blood transfusion.
This study highlight on the riskiness of transfuse blood contain integrated HBV DNA owing to the carcinogenic effect of integrated HBV in human liver. Wide-national study is required to ensure blood transfusion safety and effectiveness by using traditional (surface antigen, core antigen as a serological test) confirmed by NAT testing to screen blood donors and introduce new techniques for the detection of viral integrated with the human genome.