Mutational Events in Consecutive Passages of Hepatitis A Virus Strain GBM during Cell Culture Adaptation

Graff, Judith; Kasang, Christa; Normann, Andrea; Pfisterer-Hunt, Mechtild; Feinstone, Stephen M.; Flehmig, Bertram

doi:10.1006/viro.1994.1510

Cited by 50 publications

(46 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Adaptation of a virus to non-natural host cells is often accompanied by the attenuation of the virulence against natural host animals, as has been reported with many viruses such as influenza virus (Brown, 1990), Japanese encephalitis virus (Cao et al, 1995), hepatitis A virus (Graff et al, 1994), Borna disease virus (Rubin et al, 1993) and human immunodeficiency virus (Fujita et al, 1992 ;Sawyer et al, 1994). The mechanisms of attenuation\adaptation have been intensively studied to make use of them for designing new vaccines.…”

Section: Discussionmentioning

confidence: 89%

Isolation of an avirulent mutant of Sendai virus with two amino acid mutations from a highly virulent field strain through adaptation to LLC-MK2 cells.

Itoh

Isegawa

Hotta

et al. 1997

Journal of General Virology

View full text Add to dashboard Cite

A field strain of Sendai virus (SeV) Ohita-M1 (M1) was isolated from an epidemic in an animal laboratory by passaging in mice. A mutant strain, Ohita-MVC11 (MVC11), was then obtained by passaging M1 in rhesus monkey (LLC-MK 2 ) cells. MVC11 was adapted to LLC-MK 2 cells and produced 20 times higher levels of infectious virus than M1. This increased production of infectious virus in LLC-MK 2 cells was associated with enhanced viral gene expression. However, MVC11 could not replicate efficiently in mouse lung and was not lethal to mice even when inoculated at a titre of 8i10 5 cell-

show abstract

Section: Discussionmentioning

confidence: 89%

Isolation of an avirulent mutant of Sendai virus with two amino acid mutations from a highly virulent field strain through adaptation to LLC-MK2 cells.

Itoh

Isegawa

Hotta

et al. 1997

Journal of General Virology

View full text Add to dashboard Cite

show abstract

“…The creation of a single mutation within the 2B-coding region partially but not completely restored the replication capacity of the RNA. This mutation at nt 3889 has been identified in several independently isolated cell culture-adapted strains of HAV (9,21,24). Because the 2B protein is likely to be part of the HAV replicase complex, it has been assumed to play a role in adapting the replicase to function in a novel cellular environment.…”

Section: Discussionmentioning

confidence: 99%

“…However, certain mutations in the 2B and 2C proteins appear to be most important for replication of the virus in cultured cells. In particular, a C-to-T substitution at nucleotide (nt) 3889 that encodes an Ala-to-Val substitution at residue 1052 of the polyprotein (2B) has been identified in several different cell culture-adapted HAV variants (9,12,21,24).…”

mentioning

confidence: 99%

“…The fact that mutations in the 2B and 2C proteins enhance the growth of virus suggests indirectly that the synthesis of new copies of viral RNA may be restricted in cultured cells (9,12,21,24). However, other than the location of these mutations, there is no direct evidence that supports this hypothesis over a restriction at some other step in the viral life cycle.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Lemon

2002

J Virol

View full text Add to dashboard Cite

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 3D pol (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.

show abstract

“…Host-mediated (HM) mutations have been particularly well studied in the influenza A virus (1). However, this phenomenon has been documented in many other viruses, such as HIV, Japanese encephalitis virus, hepatitis A, and Sendai virus as well (2)(3)(4)(5). Molecular evolution studies using such sequences thus risk drawing inferences about the adaptation of the pathogen to its natural host from data containing laboratory artifacts.…”

mentioning

confidence: 99%

Effects of passage history and sampling bias on phylogenetic reconstruction of human influenza A evolution

Bush

Smith²,

Cox³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

In this paper we determine the extent to which host-mediated mutations and a known sampling bias affect evolutionary studies of human influenza A. Previous phylogenetic reconstruction of influenza A (H3N2) evolution using the hemagglutinin gene revealed an excess of nonsilent substitutions assigned to the terminal branches of the tree. We investigate two hypotheses to explain this observation. The first hypothesis is that the excess reflects mutations that were either not present or were at low frequency in the viral sample isolated from its human host, and that these mutations increased in frequency during passage of the virus in embryonated eggs. A set of 22 codons known to undergo such ''host-mediated'' mutations showed a significant excess of mutations assigned to branches attaching sequences from egg-cultured (as opposed to cell-cultured) isolates to the tree. Our second hypothesis is that the remaining excess results from sampling bias. Influenza surveillance is purposefully biased toward sequencing antigenically dissimilar strains in an effort to identify new variants that may signal the need to update the vaccine. This bias produces an excess of mutations assigned to terminal branches simply because an isolate with no close relatives is by definition attached to the tree by a relatively long branch. Simulations show that the magnitude of excess mutations we observed in the hemagglutinin tree is consistent with expectations based on our sampling protocol. Sampling bias does not affect inferences about evolution drawn from phylogenetic analyses. However, if possible, the excess caused by host-mediated mutations should be removed from studies of the evolution of influenza viruses as they replicate in their human hosts.

show abstract

Mutational Events in Consecutive Passages of Hepatitis A Virus Strain GBM during Cell Culture Adaptation

Cited by 50 publications

References 0 publications

Isolation of an avirulent mutant of Sendai virus with two amino acid mutations from a highly virulent field strain through adaptation to LLC-MK2 cells.

Isolation of an avirulent mutant of Sendai virus with two amino acid mutations from a highly virulent field strain through adaptation to LLC-MK2 cells.

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

Effects of passage history and sampling bias on phylogenetic reconstruction of human influenza A evolution

Contact Info

Product

Resources

About