Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1 [Genetic Diversity and Evolution]
Agarwal, S., Baccam, P., Aggarwal, R., Veerapu, N. S.
The American Society for Microbiology (ASM)
Published 2018
The American Society for Microbiology (ASM)
Published 2018
| Publication Date: |
2018-01-31
|
|---|---|
| Publisher: |
The American Society for Microbiology (ASM)
|
| Print ISSN: |
0022-538X
|
| Electronic ISSN: |
1098-5514
|
| Topics: |
Medicine
|
| Published by: |
| _version_ | 1836398768940908545 |
|---|---|
| autor | Agarwal, S., Baccam, P., Aggarwal, R., Veerapu, N. S. |
| beschreibung | Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 ( ORF1 ) or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10 –3 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases. IMPORTANCE Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud in vitro . We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud which had an extremely high genetic diversity had a replication rate exceeding that of wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses. |
| citation_standardnr | 6150244 |
| datenlieferant | ipn_articles |
| feed_id | 2375 |
| feed_publisher | The American Society for Microbiology (ASM) |
| feed_publisher_url | http://www.asm.org/ |
| insertion_date | 2018-01-31 |
| journaleissn | 1098-5514 |
| journalissn | 0022-538X |
| publikationsjahr_anzeige | 2018 |
| publikationsjahr_facette | 2018 |
| publikationsjahr_intervall | 7984:2015-2019 |
| publikationsjahr_sort | 2018 |
| publisher | The American Society for Microbiology (ASM) |
| quelle | Journal of Virology |
| relation | http://jvi.asm.org/cgi/content/short/92/4/e01932-17?rss=1 |
| search_space | articles |
| shingle_author_1 | Agarwal, S., Baccam, P., Aggarwal, R., Veerapu, N. S. |
| shingle_author_2 | Agarwal, S., Baccam, P., Aggarwal, R., Veerapu, N. S. |
| shingle_author_3 | Agarwal, S., Baccam, P., Aggarwal, R., Veerapu, N. S. |
| shingle_author_4 | Agarwal, S., Baccam, P., Aggarwal, R., Veerapu, N. S. |
| shingle_catch_all_1 | Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1 [Genetic Diversity and Evolution] Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 ( ORF1 ) or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10 –3 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases. IMPORTANCE Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud in vitro . We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud which had an extremely high genetic diversity had a replication rate exceeding that of wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses. Agarwal, S., Baccam, P., Aggarwal, R., Veerapu, N. S. The American Society for Microbiology (ASM) 0022-538X 0022538X 1098-5514 10985514 |
| shingle_catch_all_2 | Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1 [Genetic Diversity and Evolution] Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 ( ORF1 ) or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10 –3 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases. IMPORTANCE Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud in vitro . We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud which had an extremely high genetic diversity had a replication rate exceeding that of wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses. Agarwal, S., Baccam, P., Aggarwal, R., Veerapu, N. S. The American Society for Microbiology (ASM) 0022-538X 0022538X 1098-5514 10985514 |
| shingle_catch_all_3 | Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1 [Genetic Diversity and Evolution] Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 ( ORF1 ) or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10 –3 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases. IMPORTANCE Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud in vitro . We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud which had an extremely high genetic diversity had a replication rate exceeding that of wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses. Agarwal, S., Baccam, P., Aggarwal, R., Veerapu, N. S. The American Society for Microbiology (ASM) 0022-538X 0022538X 1098-5514 10985514 |
| shingle_catch_all_4 | Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1 [Genetic Diversity and Evolution] Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 ( ORF1 ) or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10 –3 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases. IMPORTANCE Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud in vitro . We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud which had an extremely high genetic diversity had a replication rate exceeding that of wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses. Agarwal, S., Baccam, P., Aggarwal, R., Veerapu, N. S. The American Society for Microbiology (ASM) 0022-538X 0022538X 1098-5514 10985514 |
| shingle_title_1 | Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1 [Genetic Diversity and Evolution] |
| shingle_title_2 | Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1 [Genetic Diversity and Evolution] |
| shingle_title_3 | Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1 [Genetic Diversity and Evolution] |
| shingle_title_4 | Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1 [Genetic Diversity and Evolution] |
| timestamp | 2025-06-30T23:32:20.524Z |
| titel | Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1 [Genetic Diversity and Evolution] |
| titel_suche | Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1 [Genetic Diversity and Evolution] |
| topic | WW-YZ |
| uid | ipn_articles_6150244 |