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Hepatitis B surface antigen expression: A pilot study comparing wild type and surface antigen mutant viruses
Abstracts / Journal of Clinical Virology 82S (2016) S1–S142
ples previously taken from patients presently under investigation for HEV antibodies. Materials and methods: Some patients have blood samples taken earlier in life for other routine diagnostics than HEV, and therefore it is possible to compare the former and the present sample, for determination of the HEV status of the patient, and to conclude if it is a new infection or a reactivation. We are planning to test approximately 20–30 patients all having a present HEV sample and a sample taken earlier in life for other reasons than HEV. All samples will be tested in three different commercial HEV elisa assays, one from Wantai, one from Mikrogen and one from DSI/Abia, and the results will be compared. Clinical information is available for some patients and we will try to retrieve it for the rest of the patients. Conclusion: The project is ongoing, but the final data will be ready for the conference. Reference [1] P. Grewal, S. Kamili, D. Motamed, Chronic hepatitis E in an immunocompetent patient: a case report, Hepatology 59 (1) (2014) 347–348. [2] N. Kamar, J. Izopet, Does chronic hepatitis E virus infection exist in immunocompetent patients? Hepatology (2013).
http://dx.doi.org/10.1016/j.jcv.2016.08.160 Abstract no: 342 Presentation at ESCV 2016: Poster 121 Hepatitis B surface antigen expression: A pilot study comparing wild type and surface antigen mutant viruses
S81
Preliminary results: Using more DNA in the transfection resulted in higher readouts from the ELISA. For the 0.5 m DNA transfections, two of the mutant viruses demonstrated HBsAg secretion deficiency compared with wild type virus. This occurred in one of the G145R HBV mutants and the construct with T118K, P120S and M133T surface mutations, both of which had higher HBsAg levels in the cell pellet than the supernatant fluid. The two other viruses appeared to behave like wild type virus. For the 2.0 m DNA transfections, however, these differences in secretion were not reflected, with samples demonstrating higher amounts of HBsAg in the cell pellet than the supernatant fluid. Immunofluorescence showed variation in surface antigen labelling within the HepG2 cells. The cells transfected with one of the G145R mutant viruses demonstrated greater labelling within cells than those transfected with wild type and the other mutant viruses. Conclusions and further work: The preliminary results of this pilot work indicate the presence of phenotypic differences between hepatitis B viruses with surface gene mutations. These mutations appear to have varying effects on the secretion of HBsAg from transfected cells. However, it is difficult to characterise these differences because of the contradictory results when higher amounts of DNA are used for transfection. The reasons behind the discrepancy are not clear and further work to look at the reproducibility of this phenomenon is needed. Further phenotyping work using a Luminex® bead based assay looking at variations in specific epitopes of the HBsAg will be undertaken.
Reference
A. Jeffery-Smith ∗ , J. Poh, S. Ijaz, R. Tedder Blood Born Virus Unit, Colindale, Public Health England, UK Introduction: The immunogenic region of the hepatitis B surface antigen (HBsAg), the ‘a’ determinant, is formed from a sequence of amino acids, which through disulphide bonding form a three dimensional structure. Mutations in this coding region can lead to amino acid substitutions resulting in a conformational change in the protein, which may render it unrecognisable to immunoglobulins. By creating a construct containing the PreS1/PreS2/S region of the hepatitis B virus (HBV) genome coding for the three envelope proteins the effects of mutations in the S gene on the expression of HBsAg from transfected cells can be investigated [1]. Materials and methods: Five HBV samples were investigated: a wild type, two G145R HBsAg mutants, and two with T118K, P120S and M133T, and M133T, P135L, G145R and V168A surface mutations respectively. Nested PCR was used to obtain a 0.7x HBV genome (0.7mer) construct containing the PreS1/PreS2/S coding region. The TOPO-TA cloning kit (ThermoFisher) was used to clone the constructs prior to transferring them into a mammalian expression vector. Chemical transfection of HepG2 cells was performed with the FuGENE® HD transfection system (Promega® ) using both 0.5 m and 2.0 m of DNA of the recombinant expression vector. Cells were harvested 72 h post-transfection. Supernatant fluid and cell pellets were collected. Cells grown on coverslips were fixed and labelled for microscopy. Enzyme-linked immunosorbent assay (ELISA) for HBsAg (Murex) was used to detect HBsAg in cell pellets and supernatant fluid. Coverslips were labelled with antibodies to HBsAg, golgi and nuclear proteins for confocal microscopy.
[1] T. Garcia, et al., Drastic reduction in the production of subviral particles does not impair hepatitis B virus virion secretion, J. Virol. (2009).
http://dx.doi.org/10.1016/j.jcv.2016.08.161 Abstract no: 35 Presentation at ESCV 2016: Poster 122 Comparison of immunoassays from three chemiluminescent automated systems for the detection of hepatitis B virus serological markers J. Lis-Tønder ∗ , A. Løvig, G.T. Schouborg Department of Clinical Microbiology, Lillebaelt Hospital, Vejle, Denmark Background: HBV infection is a serious global health problem. More than 350 million people suffering from chronic infection what results in 500,000 to 1.2 million deaths per year [1]. In Denmark the disease is relatively rare – the prevalence in the adult population estimated per 31st December 2007 was 0.24% [2]. Serological markers of hepatitis B virus (HBV) are used for laboratory diagnosis and monitoring of HBV infection or immune status. Hepatitis B surface antigen (HBsAg) is the hallmark of HBV infection and is the first serological marker detectable in serum, while antibodies to HBsAg (anti-HBs) can be formed following a hepatitis B infection or after hepatitis B vaccination. Hepatitis B core antibodies (IgM and IgG) are used to follow the progression of the infection from the acute stage to recovery. Anti-HBc antibodies are sometimes present after the disappearance of the HBsAg and before appearance of anti-HBs. In these situations these antibodies serve as the primary marker for infection. The hepatitis B e antigen is the marker of viral replication, and anti-HBe is a marker of immune response to HBeAg,
ples previously taken from patients presently under investigation for HEV antibodies. Materials and methods: Some patients have blood samples taken earlier in life for other routine diagnostics than HEV, and therefore it is possible to compare the former and the present sample, for determination of the HEV status of the patient, and to conclude if it is a new infection or a reactivation. We are planning to test approximately 20–30 patients all having a present HEV sample and a sample taken earlier in life for other reasons than HEV. All samples will be tested in three different commercial HEV elisa assays, one from Wantai, one from Mikrogen and one from DSI/Abia, and the results will be compared. Clinical information is available for some patients and we will try to retrieve it for the rest of the patients. Conclusion: The project is ongoing, but the final data will be ready for the conference. Reference [1] P. Grewal, S. Kamili, D. Motamed, Chronic hepatitis E in an immunocompetent patient: a case report, Hepatology 59 (1) (2014) 347–348. [2] N. Kamar, J. Izopet, Does chronic hepatitis E virus infection exist in immunocompetent patients? Hepatology (2013).
http://dx.doi.org/10.1016/j.jcv.2016.08.160 Abstract no: 342 Presentation at ESCV 2016: Poster 121 Hepatitis B surface antigen expression: A pilot study comparing wild type and surface antigen mutant viruses
S81
Preliminary results: Using more DNA in the transfection resulted in higher readouts from the ELISA. For the 0.5 m DNA transfections, two of the mutant viruses demonstrated HBsAg secretion deficiency compared with wild type virus. This occurred in one of the G145R HBV mutants and the construct with T118K, P120S and M133T surface mutations, both of which had higher HBsAg levels in the cell pellet than the supernatant fluid. The two other viruses appeared to behave like wild type virus. For the 2.0 m DNA transfections, however, these differences in secretion were not reflected, with samples demonstrating higher amounts of HBsAg in the cell pellet than the supernatant fluid. Immunofluorescence showed variation in surface antigen labelling within the HepG2 cells. The cells transfected with one of the G145R mutant viruses demonstrated greater labelling within cells than those transfected with wild type and the other mutant viruses. Conclusions and further work: The preliminary results of this pilot work indicate the presence of phenotypic differences between hepatitis B viruses with surface gene mutations. These mutations appear to have varying effects on the secretion of HBsAg from transfected cells. However, it is difficult to characterise these differences because of the contradictory results when higher amounts of DNA are used for transfection. The reasons behind the discrepancy are not clear and further work to look at the reproducibility of this phenomenon is needed. Further phenotyping work using a Luminex® bead based assay looking at variations in specific epitopes of the HBsAg will be undertaken.
Reference
A. Jeffery-Smith ∗ , J. Poh, S. Ijaz, R. Tedder Blood Born Virus Unit, Colindale, Public Health England, UK Introduction: The immunogenic region of the hepatitis B surface antigen (HBsAg), the ‘a’ determinant, is formed from a sequence of amino acids, which through disulphide bonding form a three dimensional structure. Mutations in this coding region can lead to amino acid substitutions resulting in a conformational change in the protein, which may render it unrecognisable to immunoglobulins. By creating a construct containing the PreS1/PreS2/S region of the hepatitis B virus (HBV) genome coding for the three envelope proteins the effects of mutations in the S gene on the expression of HBsAg from transfected cells can be investigated [1]. Materials and methods: Five HBV samples were investigated: a wild type, two G145R HBsAg mutants, and two with T118K, P120S and M133T, and M133T, P135L, G145R and V168A surface mutations respectively. Nested PCR was used to obtain a 0.7x HBV genome (0.7mer) construct containing the PreS1/PreS2/S coding region. The TOPO-TA cloning kit (ThermoFisher) was used to clone the constructs prior to transferring them into a mammalian expression vector. Chemical transfection of HepG2 cells was performed with the FuGENE® HD transfection system (Promega® ) using both 0.5 m and 2.0 m of DNA of the recombinant expression vector. Cells were harvested 72 h post-transfection. Supernatant fluid and cell pellets were collected. Cells grown on coverslips were fixed and labelled for microscopy. Enzyme-linked immunosorbent assay (ELISA) for HBsAg (Murex) was used to detect HBsAg in cell pellets and supernatant fluid. Coverslips were labelled with antibodies to HBsAg, golgi and nuclear proteins for confocal microscopy.
[1] T. Garcia, et al., Drastic reduction in the production of subviral particles does not impair hepatitis B virus virion secretion, J. Virol. (2009).
http://dx.doi.org/10.1016/j.jcv.2016.08.161 Abstract no: 35 Presentation at ESCV 2016: Poster 122 Comparison of immunoassays from three chemiluminescent automated systems for the detection of hepatitis B virus serological markers J. Lis-Tønder ∗ , A. Løvig, G.T. Schouborg Department of Clinical Microbiology, Lillebaelt Hospital, Vejle, Denmark Background: HBV infection is a serious global health problem. More than 350 million people suffering from chronic infection what results in 500,000 to 1.2 million deaths per year [1]. In Denmark the disease is relatively rare – the prevalence in the adult population estimated per 31st December 2007 was 0.24% [2]. Serological markers of hepatitis B virus (HBV) are used for laboratory diagnosis and monitoring of HBV infection or immune status. Hepatitis B surface antigen (HBsAg) is the hallmark of HBV infection and is the first serological marker detectable in serum, while antibodies to HBsAg (anti-HBs) can be formed following a hepatitis B infection or after hepatitis B vaccination. Hepatitis B core antibodies (IgM and IgG) are used to follow the progression of the infection from the acute stage to recovery. Anti-HBc antibodies are sometimes present after the disappearance of the HBsAg and before appearance of anti-HBs. In these situations these antibodies serve as the primary marker for infection. The hepatitis B e antigen is the marker of viral replication, and anti-HBe is a marker of immune response to HBeAg,