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1.
J Virol ; 88(2): 868-77, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24198420

RESUMO

Genotype 1 hepatitis E viruses (HEVs) are restricted to primate hosts, whereas genotype 3 HEVs predominantly infect swine, in addition to primates. In order to identify possible determinants of the host range, infectious recombinant viruses and chimeras of a genotype 1 isolate and a genotype 3 isolate were compared for their ability to infect versus transfect cultured human HepG2/C3A cells and swine LLC-PK cells. The patterns of luciferase expression from virus replicons containing the Gaussia luciferase gene in place of the viral ORF2 or ORF3 genes demonstrated that translation of the ORF2 capsid gene of genotype 1 virus is severely inhibited in swine kidney cells compared to its translation in rhesus macaque kidney or human liver cells. Therefore, this virus may produce insufficient capsid protein for optimal assembly in swine cells. Infectivity assays with a virus containing a chimeric capsid protein confirmed that amino acids 456 to 605 of the virus capsid protein comprised the virus receptor-binding region and suggested that genotype 1 viruses may be prevented from infecting swine because genotype 1 viruses are unable to enter swine cells. Rhesus macaque cells appeared to be better than human cells for growing the genotype 1 virus. These cell and virus combinations may serve as a useful in vitro model with which to study determinants of the natural host range of this virus.


Assuntos
Vírus da Hepatite E/fisiologia , Hepatite E/virologia , Hepatite Animal/virologia , Rim/virologia , Doenças dos Suínos/virologia , Animais , Linhagem Celular , Genótipo , Vírus da Hepatite E/classificação , Vírus da Hepatite E/genética , Vírus da Hepatite E/isolamento & purificação , Especificidade de Hospedeiro , Humanos , Macaca mulatta , Suínos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
2.
J Virol ; 86(10): 5697-707, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22398290

RESUMO

An infectious cDNA clone of a genotype 3 strain of hepatitis E virus adapted to growth in HepG2/C3A human hepatoma cells was constructed. This virus was unusual in that the hypervariable region of the adapted virus contained a 171-nucleotide insertion that encoded 58 amino acids of human S17 ribosomal protein. Analyses of virus from six serial passages indicated that genomes with this insert, although initially rare, were selected during the first passage, suggesting it conferred a significant growth advantage. RNA transcripts from this cDNA and the viruses encoded by them were infectious for cells of both human and swine origin, the major host species for this zoonotic virus. Mutagenesis studies demonstrated that the S17 insert was a major factor in cell culture adaptation. Introduction of 54 synonymous mutations into the insert had no detectable effect, thus implicating protein, rather than RNA, as the important component. Truncation of the insert by 50% decreased the levels of successful transfection by ~3-fold. Substitution of the S17 sequence by a different ribosomal protein sequence or by GTPase-activating protein sequence resulted in a partial enhancement of transfection levels, whereas substitution with 58 amino acids of green fluorescent protein had no effect. Therefore, both the sequence length and the amino acid composition of the insert were important. The S17 sequence did not affect transfection of human hepatoma cells when inserted into the hypervariable region of a genotype 1 strain, but this chimeric genome acquired a dramatic ability to replicate in hamster cells.


Assuntos
Vírus da Hepatite E/crescimento & desenvolvimento , Vírus da Hepatite E/genética , Hepatite E/genética , Hepatite E/virologia , Mutagênese Insercional , Recombinação Genética , Proteínas Ribossômicas/genética , Animais , Linhagem Celular Tumoral , Genótipo , Hepatite E/metabolismo , Vírus da Hepatite E/classificação , Vírus da Hepatite E/isolamento & purificação , Humanos , Dados de Sequência Molecular , Proteínas Ribossômicas/metabolismo , Suínos
3.
J Gen Virol ; 93(Pt 3): 526-530, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22113007

RESUMO

Hepatitis E virus is the aetiological agent of acute hepatitis E, a self-limiting disease prevalent in developing countries. Molecular analysis of viral genomic RNA from a chronically infected patient confirmed the recent discovery that chronic infection correlated with extensive diversification of the virus quasispecies: the hypervariable region of some virus genomes in this USA patient contained large continuous deletions and a minor proportion of genomes in faeces and serum had acquired a mammalian sequence that encoded 39 aa of S19 ribosomal protein fused to the virus non-structural protein. Genomes with this insert were selected during virus passage in cultured cells to become the predominant species, suggesting that the inserted sequence promoted virus growth. The results demonstrated that hepatitis E virus can mutate dramatically during a prolonged infection and suggests it may be important to prevent or cure chronic infections before new variants with unpredictable properties arise.


Assuntos
Fezes/virologia , Vírus da Hepatite E/crescimento & desenvolvimento , Vírus da Hepatite E/isolamento & purificação , Hepatite E/virologia , Hepatite Crônica/virologia , Soro/virologia , Carga Viral , Sequência de Aminoácidos , Linhagem Celular , Hepatócitos/virologia , Humanos , Dados de Sequência Molecular , Mutagênese Insercional , RNA Viral/genética , Recombinação Genética , Análise de Sequência de DNA , Deleção de Sequência , Estados Unidos , Cultura de Vírus
4.
Hum Mol Genet ; 9(14): 2141-7, 2000 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10958653

RESUMO

We have identified a mutation in the myotilin gene in a large North American family of German descent expressing an autosomal dominant form of limb girdle muscular dystrophy (LGMD1A). We have previously mapped this gene to 5q31. Symptoms of this adult onset disease are progressive weakness of the hip and shoulder girdles, as well as a distinctive dysarthric pattern of speech. Muscle of affected individuals shows degeneration of myofibers, variations in fiber size, fiber splitting, centrally located myonuclei and a large number of autophagic vesicles. Affected muscle also exhibits disorganization and streaming of the Z-line similar to that seen in nemaline myopathy. We have identified a C450T missense mutation in the myotilin gene that is predicted to result in the conversion of residue 57 from threonine to isoleucine. This mutation has not been found in 396 control chromosomes. The mutant allele is transcribed and normal levels of correctly localized myotilin protein are seen in LGMD1A muscle. Myotilin is a sarcomeric protein that binds to alpha-actinin and is localized in the Z-line. The observed missense mutation does not disrupt binding to alpha-actinin.


Assuntos
Proteínas Musculares/genética , Distrofias Musculares/genética , Mutação , Actinina/metabolismo , Adulto , Alelos , Sequência de Aminoácidos , Animais , Western Blotting , Núcleo Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Cromossomos Humanos Par 5 , Conectina , Sequência Conservada , Proteínas do Citoesqueleto , Etiquetas de Sequências Expressas , Feminino , Genes Dominantes , Humanos , Imuno-Histoquímica , Isoleucina/genética , Masculino , Camundongos , Proteínas dos Microfilamentos , Microscopia Eletrônica , Dados de Sequência Molecular , Proteínas Musculares/metabolismo , Proteínas Musculares/ultraestrutura , Mutação de Sentido Incorreto , Polimorfismo Conformacional de Fita Simples , Ligação Proteica , Análise de Sequência de DNA , Treonina/genética , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido
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