Determinants in 3Dpol Modulate the Rate of Growth of Hepatitis A Virus

ABSTRACT Hepatitis A virus (HAV), an atypical member of the Picornaviridae, grows poorly in cell culture. To define determinants of HAV growth, we introduced a blasticidin (Bsd) resistance gene into the virus genome and selected variants that grew at high concentrations of Bsd. The mutants grew fast and had increased rates of RNA replication and translation but did not produce significantly higher virus yields. Nucleotide sequence analysis and reverse genetic studies revealed that a T6069G change resulting in a F42L amino acid substitution in the viral polymerase (3Dpol) was required for growth at high Bsd concentrations whereas a silent C7027T mutation enhanced the growth rate. Here, we identified a novel determinant(s) in 3Dpol that controls the kinetics of HAV growth.

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5478746 cDNA encoding attenuated cell culture adapted hepatitis a virus genome

Biotechnology Advances ◽

10.1016/s0734-9750(97)88472-8 ◽

1997 ◽

Vol 15 (1) ◽

pp. 247-248

Keyword(s):

Cell Culture ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Virus Genome

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Effectiveness of Commercial Disinfectants for Inactivating Hepatitis A Virus on Agri-Food Surfaces

Journal of Food Protection ◽

10.4315/0362-028x-66.1.115 ◽

2003 ◽

Vol 66 (1) ◽

pp. 115-119 ◽

Cited By ~ 24

Author(s):

J. JEAN ◽

J.-F. VACHON ◽

O. MORONI ◽

A. DARVEAU ◽

I. KUKAVICA-IBRULJ ◽

...

Keyword(s):

Active Ingredient ◽

Quaternary Ammonium ◽

High Density Polyethylene ◽

Contact Time ◽

Sulfonic Acid ◽

Hepatitis A ◽

Incubation Temperature ◽

Hepatitis A Virus ◽

Dodecylbenzene Sulfonic Acid ◽

High Concentrations

Six commercial disinfectants were tested for their efficacy in inactivating hepatitis A virus in solution or attached to agri-food surfaces. Disinfectant I contains 10% quaternary ammonium plus 5% glutaraldehyde;disinfectant II contains 12% sodium hypochlorite; disinfectant III contains 2.9% dodecylbenzene sulfonic acid plus 16% phosphoric acid; disinfectant IV contains 10% quaternary ammonium; disinfectant V contains 2% iodide; and disinfectant VI contains 2% stabilized chlorine dioxide. Among these, disinfectants I and II were shown to be the most effective in inactivating hepatitis A virus in solution. The efficacy of these disinfectants was further tested against hepatitis A virus attached to common agri-food surfaces, including polyvinyl chlorine, high-density polyethylene, aluminum, stainless steel, and copper. Disinfectant II was shown to be the most effective, with a maximum inactivation level of about 3 log10. The inactivation efficacy was shown to be affected by the concentration of the active ingredient, the contact time between the disinfectant and the contaminated surfaces, and the incubation temperature. In general, hepatitis A virus was shown to be highly resistant to most disinfectants tested, and high concentrations of active ingredient were needed to achieve acceptable inactivation levels.

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Thermal Inactivation Kinetics of Human Norovirus Surrogates and Hepatitis A Virus in Turkey Deli Meat

Applied and Environmental Microbiology ◽

10.1128/aem.00874-15 ◽

2015 ◽

Vol 81 (14) ◽

pp. 4850-4859 ◽

Cited By ~ 10

Author(s):

Hayriye Bozkurt ◽

Doris H. D'Souza ◽

P. Michael Davidson

Keyword(s):

Hepatitis A ◽

Thermal Inactivation ◽

Hepatitis A Virus ◽

Weibull Model ◽

Activation Energies ◽

Inactivation Kinetics ◽

Order Model ◽

Decimal Reduction Time ◽

First Order ◽

Kinetics Of

ABSTRACTHuman noroviruses (HNoV) and hepatitis A virus (HAV) have been implicated in outbreaks linked to the consumption of presliced ready-to-eat deli meats. The objectives of this research were to determine the thermal inactivation kinetics of HNoV surrogates (murine norovirus 1 [MNV-1] and feline calicivirus strain F9 [FCV-F9]) and HAV in turkey deli meat, compare first-order and Weibull models to describe the data, and calculate Arrhenius activation energy values for each model. TheD(decimal reduction time) values in the temperature range of 50 to 72°C calculated from the first-order model were 0.1 ± 0.0 to 9.9 ± 3.9 min for FCV-F9, 0.2 ± 0.0 to 21.0 ± 0.8 min for MNV-1, and 1.0 ± 0.1 to 42.0 ± 5.6 min for HAV. Using the Weibull model, thetD = 1(time to destroy 1 log) values for FCV-F9, MNV-1, and HAV at the same temperatures ranged from 0.1 ± 0.0 to 11.9 ± 5.1 min, from 0.3 ± 0.1 to 17.8 ± 1.8 min, and from 0.6 ± 0.3 to 25.9 ± 3.7 min, respectively. Thez(thermal resistance) values for FCV-F9, MNV-1, and HAV were 11.3 ± 2.1°C, 11.0 ± 1.6°C, and 13.4 ± 2.6°C, respectively, using the Weibull model. Thezvalues using the first-order model were 11.9 ± 1.0°C, 10.9 ± 1.3°C, and 12.8 ± 1.7°C for FCV-F9, MNV-1, and HAV, respectively. For the Weibull model, estimated activation energies for FCV-F9, MNV-1, and HAV were 214 ± 28, 242 ± 36, and 154 ± 19 kJ/mole, respectively, while the calculated activation energies for the first-order model were 181 ± 16, 196 ± 5, and 167 ± 9 kJ/mole, respectively. Precise information on the thermal inactivation of HNoV surrogates and HAV in turkey deli meat was generated. This provided calculations of parameters for more-reliable thermal processes to inactivate viruses in contaminated presliced ready-to-eat deli meats and thus to reduce the risk of foodborne illness outbreaks.

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Replication of Subgenomic Hepatitis A Virus RNAs Expressing Firefly Luciferase Is Enhanced by Mutations Associated with Adaptation of Virus to Growth in Cultured Cells

Journal of Virology ◽

10.1128/jvi.76.3.1171-1180.2002 ◽

2002 ◽

Vol 76 (3) ◽

pp. 1171-1180 ◽

Cited By ~ 35

Author(s):

MinKyung Yi ◽

Stanley M. Lemon

Keyword(s):

Cell Culture ◽

Hepatitis A ◽

Cultured Cells ◽

Hepatitis A Virus ◽

Internal Ribosomal Entry Site ◽

Firefly Luciferase ◽

Virus Genome ◽

Wild Type Virus ◽

Reporter System ◽

Entry Site

ABSTRACT Replication of hepatitis A virus (HAV) in cultured cells is inefficient and difficult to study due to its protracted and generally noncytopathic cycle. To gain a better understanding of the mechanisms involved, we constructed a subgenomic HAV replicon by replacing most of the P1 capsid-coding sequence from an infectious cDNA copy of the cell culture-adapted HM175/18f virus genome with sequence encoding firefly luciferase. Replication of this RNA in transfected Huh-7 cells (derived from a human hepatocellular carcinoma) led to increased expression of luciferase relative to that in cells transfected with similar RNA transcripts containing a lethal premature termination mutation in 3Dpol (RNA polymerase). However, replication could not be confirmed in either FrhK4 cells or BSC-1 cells, cells that are typically used for propagation of HAV. Replication was substantially slower than that observed with replicons derived from other picornaviruses, as the basal luciferase activity produced by translation of input RNA did not begin to increase until 24 to 48 h after transfection. Replication of the RNA was reversibly inhibited by guanidine. The inclusion of VP4 sequence downstream of the viral internal ribosomal entry site had no effect on the basal level of luciferase or subsequent increases in luciferase related to its amplification. Thus, in this system this sequence does not contribute to viral translation or replication, as suggested previously. Amplification of the replicon RNA was profoundly enhanced by the inclusion of P2 (but not 5′ noncoding sequence or P3) segment mutations associated with adaptation of wild-type virus to growth in cell culture. These results provide a simple reporter system for monitoring the translation and replication of HAV RNA and show that critical mutations that enhance the growth of virus in cultured cells do so by promoting replication of viral RNA in the absence of encapsidation, packaging, and cellular export of the viral genome.

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