Hepatitis B virus (HBV) is known to be highly infective and 0 with long-term morbidity and mortality due to complications like cirrhosis, portal hypertension and hepatocellular carcinoma. HBV is a DNA virus classified in the virus family Hepadnaviridae. Humans are the only known natural host. HBV enters the liver via the bloodstream, and replication occurs only in liver tissue.

1. MANGALAYATAN
UNIVERSITY
Institute of Engineering & Technology
HBV genomic DNA Genotyping
PRESENTED BY: SUGAM BIRTHARE
B. Tech (BT)
sugam.bir@gmail.com

2. Introduction of HBV
 Hepatitis B virus (HBV) is a partially double stranded DNA
containing virus of Hepadnaviridae family . Humans are the only
known natural host.
 HBV enters the liver via the bloodstream, and replication occurs
only in liver tissue. The intact, infectious virus is 42–47 nm in
diameter and circulates in the blood in concentrations as high as
108 virions per ml.
 HBeAg partially double-stranded 3,200-nucleotide DNA
molecule, and DNA polymerase with reverse transcriptase
activity.
 HBV cause the Hepatitis B which may be acute or chronic in
nature.

3. Structure of HBV

4. Transmission Of hepatitis B viruses
 Hepatitis B is largely transmitted through exposure to bodily
fluids containing the virus.
 This includes unprotected sexual contact, blood transfusion,
re-use of contaminated needles and syringes, vertical
transmission from mother to child during childbirth, and so
on.
 The risk of transmission is closely related to the rate of Viral
replication in the infected individual at the time of the
exposure.
 hepatitis B viruses cannot spread by casual contact, such as
holding hand sharing eating utensils or drinking glasses breast
feeding, kissing, hugging, coughing or sneezing.

5. Diagnosis of HBV
 The following tests are done to identify and monitor liver
damage from hepatitis B:
I. Albumin level
II. Liver function tests
III. Prothrombin time
 The following tests are done to help diagnose and monitor
people with hepatitis B:
I. Antibody to HBsAg (Anti-HBs)
II. Antibody to hepatitis B core antigen (Anti-HBc)
III. Hepatitis B surface antigen (HBsAg)
IV. Hepatitis E surface antigen (HBeAg)

6. History
 Worldwide, two billion people have been infected with hepatitis
B virus (HBV), 360 million have chronic infection current
trends, policies, and directions. Worldwide, two billion people
have been infected with hepatitis B virus (HBV), 360 million
have chronic infection current trends, policies, and directions.
 Vaccination and 600,000 die each year from HBV-related liver
disease or hepatocellular carcinoma.
 In the 1990s, many industrialized countries and a few less-
developed countries implemented universal hepatitis B
immunization and experienced measurable reductions in HBV-
related disease. For example, in Taiwan, the prevalence of
chronic infection in children declined by more than 90%.

7. Epidemiology in India
 India is in the intermediate zone of endemicity with a
prevalence of 4.7% and contributing to 10-15% of the total
infected population, worldwide.
 In India, 2,50,000 infants get infected every year and 90%
of then, develop chronic infection.
 Professional blood donors constitute the major high-risk
group for HBV infection in India, with a hepatitis B
surface antigen positivity rate of 14%.

8. Sign and Symptoms
 Acute infection with hepatitis B virus is associated with
acute viral hepatitis B virus: an illness that begins with
general ill-health, loss of appetite, nausea, vomiting, body
aches, mild fever, and dark urine, and then progresses to
development of jaundice.
 Chronic infection with hepatitis B virus: This type of
infection dramatically increases the incidence
of hepatocellular carcinoma (liver cancer), liver damage
(cirrhosis), death.

9. Hepatitis B Virus Genotyping
 HBV has been classified into 6 genotypes (A-F) based on intergroup
divergence of 8% or more in the complete nucleotide sequence.
 Most of the information today is based on studies of patients with
chronic HBV infection in Asia.
 Studies from India have generally reported predominance of
genotype D.
 This raises the possibility that the Indian population originally had
genotype D, which has been replaced by genotype A particularly in
northern India due to human migration from Europe over time.
 The study also showed that patients with genotype F were more
likely to die from liver disease than those with A and D.

10. Extraction of nucleic acid for HBV genotyping
through High Pure System nucleic acid
extraction kit (Roche):
 Opening one well at a time, pipette 200 μl of specimen or control into the
appropriate well.*
 Pipette 200 μl of Lysis Solution and 40 μl Proteinase K into each well of
the Lysis Rack (I,white or transparent).*
 After all specimens and controls have been added, mix by vortexing the
filled Lysis Rack for approximately 10 seconds.
 Incubate the Lysis Rack in a preheated 50°C (± 2°C) water bath for 15
minutes.
 Mix by vortexing the filled Lysis Rack for approximately 10 seconds.
 Opening one well at a time, pipette 210 μl of isopropanol into each well.*
 *Tightly close the lids.

11. Continued…
 Mix specimens by inverting the rack three times, then vortexing the rack for
approximately 10 seconds.
 Opening one well at a time, transfer whole mixture to the corresponding
wells of the Filter Tube Rack (II, yellow) with affixed Waste Rack (white).
Discard the Lysis Rack appropriately.
 Centrifuge the Filter Tube Rack assembly for 2 minutes at 4600 x g in the
micro-titer plate centrifuge.
 Open all lids of the Filter Tube Rack with the Grippers and pipette 400 μl of
Inhibitor Removal Buffer (IRB) down the side of each well.*
 Centrifuge the Filter Tube Rack assembly for 2 minutes at 4600 x g in the
micro-titer plate centrifuge.
 Open all lids with the Grippers and pipette 500 μl of Wash Buffer (WASH)
down the side of each well.*
 *Tightly close the lids.

12. Continued…
 Centrifuge the Filter Tube Rack assembly for 2 minutes at 4600 x g in the
micro-titer plate centrifuge.
 Open all lids with the Grippers and pipette 500 μl of Wash Buffer down the
side of each well.*
 Centrifuge the Filter Tube Rack assembly for 3 minutes at 4600 x g in the
micro-titer plate centrifuge.
 Remove the Filter Tube Rack from the Waste Rack by pressing both snap
links on the upper side of the Filter Tube Rack.
 Place the Filter Tube Rack onto the Elution Rack (IIIA, blue) and snap the
Filter Tube Rack onto the Elution Rack. Discard the Waste Rack
appropriately.
 Open all lids with the Grippers and pipette 75 μl of the prewarmed Elution
Buffer (ELB) onto the center of each filter without touching the filter.*
 *Tightly close the lids.

13. Continued…
 Incubate the Elution Rack at room temperature for a minimum of 3 minutes
after adding Elution Buffer to the last well.
 Place the Cover Rack (IIIB, blue) onto the Elution Rack (IIIA, blue). Press
down firmly and snap links onto the Elution Rack. Close all lids.
 The processed specimens and controls are used directly for PCR. Use 50 μl
of the processed specimens and controls for amplification.
 Add the processed specimens and controls to the Working Master Mix within
3 hours of completing specimen and control preparation. If processed
specimens and controls cannot be used within 3 hours of preparation, the
processed specimens and controls can be stored at 2-8°C for up to 24 hours
in the covered Elution Rack or frozen at -20°C for up to 1 week in sterile 2.0
ml polypropylene screw-cap tubes.
 Amplify the processed specimens and controls within 3 hours of adding the
specimens and controls to the Working Master Mix.

14. Result
 The current study includes collection of 55 blood specimen from the different
Departments of Shri Mahant Indiresh Hospital, Dehradun (U.K.).
 Serum/Plasma was collected from all the blood sample and further subjected
for different molecular parameters.
 Out of 55 specimens HBV DNA was quantified in 43 samples by the
utilization of TaqMan probes in COBAS TaqMan48 Real Time PCR and their
titer values were observed and ranges in-between from 2.33x101-
5.12x109IU/ml.
 The HBV genotypes were characterized through A-F. 43 specimen were
subjected for HBV genotyping out of which genotype D was most prevalent
and was found in 23 (30%) cases. Genotypes A, B, C, E and F was found
10(13%), 16(21%), 14(18%), 9(12%) and 5(6%) respectively.
 Type D(119bp) was most prevalent in age group between 21-40 years.
Although type A, B, C, E & F were also present.

15. Gel Pictures as Result
 NOTE- HBV genotype D-119 bp, HBV
genotype E-167 bp& HBV genotype F-97
bp
 Well 1- Target Not Detected
 Well 2- Target Not Detected
 Well 3- HBV genotype D & E
 Well 4- HBV genotype D & E
 Well 5- Target Not Detected
 Well 6- HBV genotype D & E
 Well 7- Target Not Detected
 Well 8- HBV genotype E
 Well 9- Target Not Detected
 Well 10- Target Not Detected
 Well 11- DNA ladder (50 bp)
1 2 3 4 5 6 7 8 9 10 11

16. References
 Lee. W. 1997. Hepatitis B Virus Infection. New England Journal of Medicine. 337:1733-1745.
 Huo, T., Wu, J., Lee, P., et al. 1998. Sero-clearance of hepatitis b surface antigen in chronic carriers
does not necessarily imply a good prognosis. Hepatology28:231-236. at ‘‘Hepatocellular Carcinoma:
Screening, Diagnosis, and Management,’’ a National Institutes of Health workshop, April 1.
 Colin W. Shepard, Edgar P. Simard, Lyn Finelli, Anthony E. Fiore, and Beth P. Bell. Division of Viral
Hepatitis, National Center for Infectious Diseases, Centers for Disease Control and Prevention,
Atlanta, GA. 2006 Epidemiologic Reviews;28:112-125.
 Fattovich G. Natural history and prognosis of hepatitis B. Semin Liver Dis 2003;23:47.

17. Thank You!