RESUMEN
The hepatitis E virus (HEV) is infecting over 20 million people annually with a high morbidity especially in pregnant women and immune-suppressed individuals. While HEV genotype 1 (HEV-1) infects only humans, genotype 3 (HEV-3) is zoonotic and commonly transmitted from infected animals to humans. Whereas a few reverse genetics systems enabling targeted genome manipulations exist for HEV-3, those for HEV-1 are still very limited, mainly because of inefficient cell culture replication. Here, the generation of HEV-1 strain Sar55 and HEV-3 strain 47832mc by transfecting in vitro-transcribed and capped virus genomes into different cell lines was attempted. Culture supernatants of colon-derived colorectal adenocarcinoma cell line Caco-2 contained HEV-1 and HEV-3 capable of infecting Caco-2 cells. Density gradient centrifugation analyses of culture supernatants confirmed that HEV-1 particles were quasi-enveloped in analogy to HEV-3 and that non-virion-associated capsid protein was secreted from cells. Following transfection or infection of Caco-2 cells, HEV-1 consistently reached higher titers than HEV-3 in culture supernatants, but HEV-1 generated by transfection of Caco-2 cells was unable to efficiently infect hepatoma cell lines PLC/PRF/5 or HuH7-Lunet BLR. Taken together, our results indicate that HEV-1 is able to exert a complete replication cycle in Caco-2 cells. An efficient cell culture system for this genotype will be useful for studying species tropism, but further research is required to determine the significance of HEV-1 replication in colon-derived cells.
Asunto(s)
Genotipo , Virus de la Hepatitis E , Genética Inversa , Replicación Viral , Humanos , Virus de la Hepatitis E/genética , Virus de la Hepatitis E/fisiología , Células CACO-2 , Genética Inversa/métodos , Colon/virología , Genoma Viral , Hepatitis E/virologíaRESUMEN
Rotavirus A (RVA) is the leading cause of diarrhea requiring hospitalization in children and causes over 100,000 annual deaths in Sub-Saharan Africa. In order to generate next-generation vaccines against African RVA genotypes, a reverse genetics system based on a simian rotavirus strain was utilized here to exchange the antigenic capsid proteins VP4, VP7 and VP6 with those of African human rotavirus field strains. One VP4/VP7/VP6 (genotypes G9-P[6]-I2) triple-reassortant was successfully rescued, but it replicated poorly in the first cell culture passages. However, the viral titer was enhanced upon further passaging. Whole genome sequencing of the passaged virus revealed a single point mutation (A797G), resulting in an amino acid exchange (E263G) in VP4. After introducing this mutation into the VP4-encoding plasmid, a VP4 mono-reassortant as well as the VP4/VP7/VP6 triple-reassortant replicated to high titers already in the first cell culture passage. However, the introduction of the same mutation into the VP4 of other human RVA strains did not improve the rescue of those reassortants, indicating strain specificity. The results show that specific point mutations in VP4 can substantially improve the rescue and replication of recombinant RVA reassortants in cell culture, which may be useful for the development of novel vaccine strains.
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Proteínas de la Cápside , Virus Reordenados , Rotavirus , Replicación Viral , Rotavirus/genética , Proteínas de la Cápside/genética , Humanos , Virus Reordenados/genética , Animales , Mutación , Línea Celular , Genética Inversa/métodos , Genotipo , Mutación Puntual , Infecciones por Rotavirus/virología , Genoma Viral , Antígenos Virales/genética , Antígenos Virales/inmunologíaRESUMEN
The hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. Whereas HEV genotypes 1-4 of species Paslahepevirus balayani are commonly found in humans, infections with ratHEV (species Rocahepevirus ratti) were previously considered to be restricted to rats. However, several cases of human ratHEV infections have been described recently. To investigate the zoonotic potential of this virus, a genomic clone was constructed here based on sequence data of ratHEV strain pt2, originally identified in a human patient with acute hepatitis from Hongkong. For comparison, genomic clones of ratHEV strain R63 from a rat and of HEV genotype 3 strain 47832mc from a human patient were used. After transfection of in vitro-transcribed RNA from the genomic clones into the human hepatoma cell line HuH-7-Lunet BLR, virus replication was shown for all strains by increasing genome copy numbers in cell culture supernatants. These cells developed persistent virus infections, and virus particles in the culture supernatant as well as viral antigen within the cells were demonstrated. All three generated virus strains successfully infected fresh HuH-7-Lunet BLR cells. In contrast, the human hepatoma cell lines HuH-7 and PLC/PRF/5 could only be infected with the genotype 3 strain and to a lesser extent with ratHEV strain R63. Infection of the rat-derived hepatoma cell lines clone 9, MH1C1 and H-4-II-E did not result in efficient virus replication for either strain. The results indicate that ratHEV strains from rats and humans can infect human hepatoma cells. The replication efficiency is strongly dependent on the cell line and virus strain. The investigated rat hepatoma cell lines could not be infected and other rat-derived cells should be tested in future to identify permissive cell lines from rats. The developed genomic clone can represent a useful tool for future research investigating pathogenicity and zoonotic potential of ratHEV.
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Virus de la Hepatitis E , Replicación Viral , Animales , Humanos , Ratas , Virus de la Hepatitis E/genética , Virus de la Hepatitis E/clasificación , Virus de la Hepatitis E/fisiología , Línea Celular Tumoral , Hepatitis E/virología , Genotipo , Genoma Viral , Carcinoma Hepatocelular/virología , ARN Viral/genética , Hepatocitos/virologíaRESUMEN
Heat treatment of food represents an important measure to prevent pathogen transmission. Thus far, evaluation of heat treatment processes is mainly based on data from bacteria. However, foodborne viruses have gained increasing attention during the last decades. Here, the published literature on heat stability and inactivation of human norovirus (NoV), hepatitis A virus (HAV) and hepatitis E virus (HEV) was reviewed. Data for surrogate viruses were not included. As stability assessment for foodborne viruses is often hampered by missing infectivity assays, an overview of applied methods is also presented. For NoV, molecular capsid integrity assays were mainly applied, but data from initial studies utilizing novel intestinal enteroid or zebrafish larvae assays are available now. However, these methods are still limited in applicability and sensitivity. For HAV, sufficient cell culture-based inactivation data are available, but almost exclusively for one single strain, thus limiting interpretation of the data for the wide range of field strains. For HEV, data are now available from studies using pig inoculation or cell culture. The results of the reviewed studies generally indicate that NoV, HAV and HEV possess a high heat stability. Heating at 70-72 °C for 2 min significantly reduces infectious titers, but often does not result in a >4 log10 decrease. However, heat stability greatly varied dependent on virus strain, matrix and heating regime. In addition, the applied method largely influenced the result, e.g. capsid integrity assays tend to result in higher measured stabilities than cell culture approaches. It can be concluded that the investigated foodborne viruses show a high heat stability, but can be inactivated by application of appropriate heating protocols. For HAV, suggestions for safe time/temperature combinations for specific foods can be derived from the published studies, with the limitation that they are mostly based on one strain only. Although significant improvement of infectivity assays for NoV and HEV have been made during the last years, further method development regarding sensitivity, robustness and broader applicability is important to generate more reliable heat inactivation data for these foodborne viruses in future.
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Virus de la Hepatitis A , Virus de la Hepatitis E , Norovirus , Animales , Humanos , Porcinos , Calor , Pez Cebra , Virus de la Hepatitis A/fisiología , Temperatura , Virus de la Hepatitis E/fisiología , Norovirus/fisiología , Inactivación de VirusRESUMEN
Rotavirus A (RVA) genome segments can reassort upon co-infection of target cells with two different RVA strains. However, not all reassortants are viable, which limits the ability to generate customized viruses for basic and applied research. To gain insight into the factors that restrict reassortment, we utilized reverse genetics and tested the generation of simian RVA strain SA11 reassortants carrying the human RVA strain Wa capsid proteins VP4, VP7, and VP6 in all possible combinations. VP7-Wa, VP6-Wa, and VP7/VP6-Wa reassortants were effectively rescued, but the VP4-Wa, VP4/VP7-Wa, and VP4/VP6-Wa reassortants were not viable, suggesting a limiting effect of VP4-Wa. However, a VP4/VP7/VP6-Wa triple-reassortant was successfully generated, indicating that the presence of homologous VP7 and VP6 enabled the incorporation of VP4-Wa into the SA11 backbone. The replication kinetics of the triple-reassortant and its parent strain Wa were comparable, while the replication of all other rescued reassortants was similar to SA11. Analysis of the predicted structural protein interfaces identified amino acid residues, which might influence protein interactions. Restoring the natural VP4/VP7/VP6 interactions may therefore improve the rescue of RVA reassortants by reverse genetics, which could be useful for the development of next generation RVA vaccines.
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Infecciones por Rotavirus , Rotavirus , Humanos , Rotavirus/genética , Proteínas de la Cápside/genética , Genética Inversa , Cápside/química , Antígenos ViralesRESUMEN
Infection with the tick-borne encephalitis virus (TBEV) can cause meningitis, meningoencephalitis and myelitis in humans. TBEV is an enveloped RNA virus of the family Flaviviridae, which is mostly transmitted via tick bites. However, transmission by consumption of virus-contaminated goat raw milk and goat raw milk products has also been described. Only a few methods have been reported for the detection of TBEV in food so far. Here, we compare different virus extraction methods for goat raw milk and goat raw milk cream cheese and subsequent detection of TBEV-RNA by RT-qPCR. Langat virus (LGTV), a naturally attenuated TBEV strain, was used for artificial contamination experiments. Mengovirus and the human coronavirus 229E were compared to assess their suitability to serve as internal process controls. Out of three tested extraction protocols for raw milk, sample centrifugation followed by direct RNA extraction from the aqueous interphase yielded the best results, with a recovery rate (RR) of 31.8 ± 4.9% for LGTV and a detection limit of 6.7 × 103 LGTV genome copies/ml. Out of two methods for cream cheese, treatment of the samples with TRI Reagent® and chloroform prior to RNA extraction showed the best RR of 4.7 ± 1.6% for LGTV and a detection limit of 9.4 × 104 LGTV genome copies/g. RRs of Mengovirus and LGTV were similar for both methods; therefore, Mengovirus is suggested as internal process control virus. The developed methods may be useful for screening or surveillance studies, as well as in outbreak investigations.
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Queso , Virus de la Encefalitis Transmitidos por Garrapatas , Encefalitis Transmitida por Garrapatas , Animales , Humanos , Leche , Virus de la Encefalitis Transmitidos por Garrapatas/genética , Cabras/genética , ARNRESUMEN
Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is primarily transmitted from human to human via droplets and aerosols. While transmission via contaminated surfaces is also considered possible, the overall risk of this transmission route is assumed to be low. Nevertheless, transmission through contaminated drinking glasses may pose an increased risk as the glass is in direct contact with the mouth and oral cavity. Using human coronavirus 229E (HCoV-229E) as surrogate for SARS-CoV-2, this study examined coronavirus stability on glass, inactivation by dishwashing detergents, and virus elimination by a manual glass scrubbing device. Infectious HCoV-229E was recovered from glass for 7 and 21 days of storage under daylight and dark conditions, respectively. Near complete inactivation of HCoV-229E (>4 log10 reduction) was observed after incubation with two common dishwashing detergents at room temperature for 15 s, whereas incubation at 43 °C for 60 s was necessary for a third detergent to achieve a similar titer reduction. The virus was efficiently removed from contaminated drinking glasses using a manual glass scrubbing device in accordance with German standard DIN 6653-3. The results confirm that coronaviruses are relatively stable on glass, but indicate that common manual dishwashing procedures can efficiently eliminate coronaviruses from drinking glasses.
Asunto(s)
COVID-19 , Coronavirus Humano 229E , COVID-19/prevención & control , Detergentes , Humanos , SARS-CoV-2RESUMEN
Species A rotaviruses (RVAs) are important aetiological agents of severe diarrhoea in young children. They are also widely distributed in mammals and birds, and increasing evidence indicates the possibility of zoonotic transmission of RVA strains between animals and humans. Moreover, reassortment of the eleven segments of the RVA genome can result in rapid biological changes and may influence pathogenic properties. Here, the nearly complete genome of an RVA strain from a common shrew (Sorex araneus) was sequenced, which showed high nucleotide sequence similarity to additionally determined partial sequences from common shrew RVAs but only very low identity (below 68 per cent) to RVAs from other animal species and humans. New genotypes were assigned to most genome segments of the novel common shrew RVA strain KS14/269, resulting in the genome constellation G39-P[55]-I27-R26-C22-M22-A37-N26-T26-E30-H26. Phylogenetic analyses clustered the common shrew RVAs as ancestral branches of other mammalian and avian RVAs for most of the genome segments, which is in contrast to the phylogeny of the hosts. Nevertheless, conserved sequences typical for all RVAs were identified at the 5'- and 3'- non-coding segment termini. To explore whether the common shrew RVA can exchange genetic material with other mammalian RVAs by reassortment, a reverse genetics system based on the simian RVA strain SA11 was used. However, no viable reassortants could be rescued by exchanging the VP4-, VP6-, or VP7-encoding genome segment alone or in combinations. It can be concluded that highly divergent RVAs are present in common shrews, indicating an evolution of these viruses largely separated from other mammalian and avian RVAs. The zoonotic potential of the virus seems to be low but needs to be further analysed in future.
RESUMEN
The hepatitis E virus (HEV) is an etiological agent of acute hepatitis in humans. In addition, chronic infections resulting in fatal liver cirrhosis currently emerge in immunosuppressed transplant patients. The number of notified hepatitis E cases in Germany has steeply increased in recent years. Here, genotype 3, which can be zoonotically transmitted from animals to humans, is predominant. The main reservoirs are pigs and wild boars, which show no signs of infection. In this article, the distribution of HEV in animals in Germany, possible transmission pathways, and especially the importance of food as a transmission vehicle are presented based on the current scientific literature.HEV is widely spread among domestic pigs and wild boars in Germany and the virus is mainly transmitted by direct contact or by consumption of food produced from those animals. However, if HEV RNA is detected in specific food it is often unclear whether the contained virus is still infectious or inactivated by the conditions during production. Recent studies indicate a high stability of HEV against different physicochemical conditions, whereas - among others - the virus can be efficiently inactivated by heating. Therefore, proper heating of pork meat and liver prior to consumption in general is recommended. For risk groups, avoiding shortly cured raw sausages is an additional suggestion.Further research is necessary to identify relevant risk food products, to investigate alternative transmission pathways, and to develop efficient measures in order to reduce or prevent zoonotic transmissions of the virus in future.
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Virus de la Hepatitis E , Hepatitis E , Animales , Inocuidad de los Alimentos , Alemania/epidemiología , Hepatitis E/epidemiología , Virus de la Hepatitis E/genética , Humanos , Sus scrofa , PorcinosRESUMEN
The hepatitis E virus (HEV) can cause hepatitis E in humans. Recently, the occurrence of HEV strains carrying insertions in their hypervariable genome region has been described in chronically infected patients. The insertions originate from human genes or from the HEV genome itself. Although their distinct functions are largely unknown, an involvement in efficient cell culture replication was shown for some strains. The HEV strain 47832c, originally isolated from a chronically infected transplant patient, carries a bipartite insertion composed of HEV genome duplications. Here, several mutants with deletions and substitutions of the insertion were generated and tested in cell culture. Complete deletion of the insertion abolished virus replication and even a single glycine to arginine substitution led to reduced cell culture growth. A mutant encoding a frameshift of the inserted sequence was not infectious, whereas a mutant carrying synonymous codons in this region replicated similar like the wild type. Substitution of the insertion with the S17 insertion from HEV strain Kernow C1-p6 did not result in viable virus, which might indicate strain- or cell type-specificity of the insertions. Generally, the translated amino acid sequence of the insertion, but not the RNA sequence, seems to be responsible for the observed effect.
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Genoma Viral , Virus de la Hepatitis E/genética , Hepatitis E/virología , Mutagénesis Insercional , Sistemas de Lectura Abierta/genética , Replicación Viral , Sustitución de Aminoácidos , Línea Celular , Células Cultivadas , Expresión Génica , Orden Génico , Humanos , Plásmidos/genética , ARN Viral/genéticaRESUMEN
The rotavirus species A (RVA) capsid contains the spike protein VP4, which interacts with VP6 and VP7 and is involved in cellular receptor binding. The capsid encloses the genome consisting of eleven dsRNA segments. Reassortment events can result in novel strains with changed properties. Using a plasmid-based reverse genetics system based on simian RVA strain SA11, we previously showed that the rescue of viable reassortants containing a heterologous VP4-encoding genome segment was strain-dependent. In order to unravel the reasons for the reassortment restrictions, we designed here a series of plasmids encoding chimeric VP4s. Exchange of the VP4 domains interacting with VP6 and VP7 was not sufficient for rescue of viable viruses. In contrast, the exchange of fragments encoding the receptor-binding region of VP4 resulted in virus rescue. All parent strains and the rescued reassortants replicated efficiently in MA-104 cells used for virus propagation. In contrast, replication in BSR T7/5 cells used for plasmid transfection was only efficient for the SA11 strain, whereas the rescued reassortants replicated slowly, and the parent strains failing to produce reassortants did not replicate. While future research in this area is necessary, replication in BSR T7/5 cells may be one factor that affects the rescue of RVAs.
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Proteínas de la Cápside/genética , Virus Reordenados/genética , Infecciones por Rotavirus/virología , Rotavirus/genética , Cápside/fisiología , Línea Celular , Humanos , Plásmidos/genética , Genética Inversa/métodos , Transfección/métodos , Replicación Viral/genéticaRESUMEN
The hepatitis E virus (HEV) is the causative agent of acute and chronic hepatitis in humans. Related viruses have been found in several animal species. Reverse genetics systems (RGSs), which enable the generation of infectious virus from cloned cDNA by transfection of cultured cells or intrahepatic injection into laboratory animals, have been developed for HEV genotypes 1, 3, 4, 5 and 7 as well as for avian HEV and rat HEV. However, low virus recovery rates and slow replication in cell cultures are observed for most of the HEV types. Nevertheless, the RGSs enabled the site-directed mutagenesis of single nucleotides, deletion of genome fragments, insertion of sequence tags and a marker gene as well as the generation of chimeric viruses.
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Virus de la Hepatitis E/genética , Hepatitis E/virología , Genética Inversa/métodos , Animales , Técnicas de Cultivo de Célula , Línea Celular , Genotipo , Virus de la Hepatitis E/clasificación , Virus de la Hepatitis E/inmunología , Humanos , Ratones , Mutagénesis Sitio-Dirigida , ARN Viral/genética , Replicación ViralRESUMEN
Species A rotavirus (RVA) is an important gastrointestinal pathogen that is widely distributed in humans, mammalian animals and birds. The RVA genome consists of eleven double-stranded RNA segments, enabling the generation of novel strains with new pathogenic or antigenic features by genetic reassortment. While reassortants between human and mammalian animal RVAs have been repeatedly described, data on the reassortment potential of avian RVA strains are rare. To investigate genome segment exchanges between avian and mammalian RVA strains, a plasmid-based reverse genetics strategy originally developed for the simian RVA strain SA11 was used here. All eleven genome segments of the chicken RVA strain 02V0002G3 were cloned into similar plasmids as in the SA11 system. However, in contrast to SA11, no infectious virus could be generated by transfection of the eleven 02V0002G3 plasmids into cell culture under the same conditions. In another series of experiments, each of the genome segments of 02V0002G3 was transfected together with the remaining ten genome segments of SA11. Viable mono-reassortants were only retrieved for the avian VP3 and VP4 genes. The reassortant viruses were structurally indistinguishable from their parental viruses, but grew to slightly lower titers in cell culture. The results indicate that the VP3 and VP4 genes, but not the other genes of avian RVA, can functionally substitute their mammalian homologs and create viable reassortants. Further research should focus on the reasons behind the reassortment incompatibility and on the optimization of the system for the generation of viable avian RVA rescued entirely from cloned avian RVA genome segments.
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Pollos/virología , Mamíferos/virología , Virus Reordenados/genética , Rotavirus/genética , Animales , Línea Celular , Genoma Viral , Genotipo , Filogenia , Plásmidos/genética , Genética Inversa , Infecciones por Rotavirus/virología , Transfección , Replicación ViralRESUMEN
Rotavirus A (RVA) is a major cause of gastroenteritis in humans and mammalian animals, and has also been abundantly detected in avian species. Avian RVA infection is associated with diarrhea, reduced growth and increased mortality, leading to economic losses in the poultry industry. Avian RVA forms a unique genetic clade within the whole RVA species. However, up to now, only a few full-length avian RVA genomes have been published and only a small number of avian RVA strains have been adapted to grow in cell culture for subsequent studies. Here, the four cell culture-adapted chicken RVA strains 02V0002G3, 04V0027G6, 05V0500F6 and 06V0661G1 were characterized in more detail. Transmission electron microscopy of the viruses derived from culture supernatant showed a typical triple-layered morphology of rotavirus particles; in addition, strain 06V0661G1 showed a high proportion of double-layered particles. The (nearly) complete genome sequences of the viruses were determined using next-generation sequencing (NGS). The resulting sequences were compared to full-length or partial sequences of the strains previously determined using Sanger sequencing; and a few nucleotide mismatches, some of them resulting in amino acid substitutions, were identified. The genomes of strains 02V0002G3, 04V0027G6 and 05V0500F6 were closely related to each other showing a G19-P[30]-I11-R6-C6-M7-A16-N6-T8-E10-H8 genotype constellation. Strain 06V0661G1 carries the VP4 genotype P[31] in the same genetic backbone like the other strains. However, further sequence analysis showed that the genes of this strain, especially that encoding NSP3, clustered more separately from the other strains in phylogenetic trees. The characterized cell culture-adapted chicken RVA strains may be useful for future studies investigating genetic diversity and replication of avian rotaviruses, as well as for the development of vaccines and diagnostic tools.
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Pollos/virología , Genoma Viral/genética , Infecciones por Rotavirus/virología , Rotavirus/genética , Animales , Células Cultivadas , Chlorocebus aethiops , Diarrea/virología , Gastroenteritis/virología , Genotipo , Humanos , Mamíferos/virología , Filogenia , Análisis de Secuencia/métodos , Secuenciación Completa del Genoma/métodosRESUMEN
The hepatitis E virus (HEV) causes acute and chronic hepatitis in humans. Investigation of HEV replication is hampered by the lack of broadly applicable, efficient cell culture systems and tools for site-directed mutagenesis of HEV. The cell culture-adapted genotype 3c strain 47832c, which represents a typical genotype predominantly detected in Europe, has previously been used for several basic and applied research studies. Here, a plasmid-based reverse genetics system was developed for this strain, which efficiently rescued the infectious virus without the need for in vitro RNA transcription. The cotransfection of T7 RNA polymerase-expressing BSR/T7 cells with one plasmid encoding the full-length viral genome and two helper plasmids encoding vaccinia virus capping enzymes resulted in the production of infectious HEV, which could be serially passaged on A549/D3 cells. The parental and recombinant virus exhibited similar replication kinetics. A single point mutation creating an additional restriction enzyme site could be successfully introduced into the virus genome of progeny virus, indicating that the system is suitable for site-directed mutagenesis. This system is the first plasmid-based HEV reverse genetics system, as well as the first reverse genetics system for HEV genotype 3c, and should therefore be of broad use for basic and applied HEV research.
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Human rotavirus A (RVA) causes acute gastroenteritis in infants and young children. The broad use of two vaccines, which are based on RVA strains from Europe and North America, significantly reduced rotavirus disease burden worldwide. However, a lower vaccine effectiveness is recorded in some regions of the world, such as sub-Saharan Africa, where diverse RVA strains are circulating. Here, a plasmid-based reverse genetics system was used to generate simian RVA reassortants with VP4 and VP7 proteins derived from African human RVA strains not previously adapted to cell culture. We were able to rescue 1/3 VP4 mono-reassortants, 3/3 VP7 mono-reassortants, but no VP4/VP7 double reassortant. Electron microscopy showed typical triple-layered virus particles for the rescued reassortants. All reassortants stably replicated in MA-104 cells; however, the VP4 reassortant showed significantly slower growth compared to the simian RVA or the VP7 reassortants. The results indicate that, at least in cell culture, human VP7 has a high reassortment potential, while reassortment of human VP4 from unadapted human RVA strains with simian RVA seems to be limited. The characterized reassortants may be useful for future studies investigating replication and reassortment requirements of rotaviruses as well as for the development of next generation rotavirus vaccines.
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Antígenos Virales/genética , Proteínas de la Cápside/genética , Genoma Viral , Virus Reordenados/genética , Rotavirus/genética , África , Animales , Técnicas de Cultivo de Célula , Línea Celular , Genotipo , Haplorrinos/virología , Humanos , Filogenia , Plásmidos/genética , Virus Reordenados/crecimiento & desarrollo , Genética Inversa , Rotavirus/crecimiento & desarrollo , Infecciones por Rotavirus/virología , Replicación ViralRESUMEN
We screened samples from common shrews (Sorex araneus) collected in Germany during 2004-2014 and identified 3 genetically divergent rotaviruses. Virus protein 6 sequence similarities to prototype rotaviruses were low (64.5% rotavirus A, 50.1% rotavirus C [tentative species K], 48.2% rotavirus H [tentative species L]). Shrew-associated rotaviruses might have zoonotic potential.
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Enfermedades de los Animales/epidemiología , Enfermedades de los Animales/virología , Infecciones por Rotavirus/veterinaria , Rotavirus , Musarañas/virología , Enfermedades de los Animales/historia , Animales , Genes Virales , Geografía Médica , Alemania/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento , Historia del Siglo XXI , Filogenia , ARN ViralRESUMEN
Species A rotaviruses (RVAs) are a major cause of gastroenteritis in animals and humans. Their genome consists of 11 segments of dsRNA, and reassortment events between animal and human strains can contribute to the high genetic diversity of RVAs. We used a plasmid-based reverse genetics system to investigate the reassortment potential of the genome segment encoding the viral outer capsid protein VP4, which is a major antigenic determinant, mediates viral entry and plays an important role in host cell tropism. We rescued reassortant viruses containing VP4 from porcine, bovine, bat, pheasant or chicken RVA strains in the backbone of simian strain SA11. The VP4 reassortants could be stably passaged in MA-104 cells and induced cytopathic effects. However, analysis of growth kinetics revealed marked differences in replication efficiency. Our results show that the VP4-encoding genome segment has a high reassortment potential, even between virus strains from highly divergent species. This can result in replication-competent reassortants with new genomic, growth and antigenic features.
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Proteínas de la Cápside/genética , Virus Reordenados/genética , Genética Inversa , Infecciones por Rotavirus/virología , Rotavirus/genética , Secuencia de Aminoácidos , Proteínas de la Cápside/química , Línea Celular , Genoma Viral , Humanos , Modelos Moleculares , Filogenia , Plásmidos/genética , Conformación Proteica , Genética Inversa/métodos , Rotavirus/clasificación , Replicación ViralRESUMEN
Conventional HIV gene therapy approaches are based on engineering HIV target cells that are non-permissive to viral replication. However, expansion of gene-modified HIV target cells has been limited in patients. Alternative genetic strategies focus on generating gene-modified producer cells that secrete antiviral proteins (AVPs). The secreted AVPs interfere with HIV entry, and, therefore, they extend the protection against infection to unmodified HIV target cells. Since any cell type can potentially secrete AVPs, hematopoietic and non-hematopoietic cell lineages can function as producer cells. Secretion of AVPs from non-hematopoietic cells opens the possibility of using a genetic approach for HIV prevention. Another strategy aims at modifying cytotoxic T cells to selectively target and eliminate infected cells. This review provides an overview of the different genetic approaches for HIV treatment and prevention.
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HIV entry is a highly specific and time-sensitive process that can be divided into receptor binding, coreceptor binding, and membrane fusion. Bifunctional antiviral proteins (bAVPs) exploit the multi-step nature of the HIV entry process by binding to two different extracellular targets. They are generated by expressing a fusion protein containing two entry inhibitors with a flexible linker. The resulting fusion proteins exhibit exceptional neutralization potency and broad cross-clade inhibition. In this review, we summarize the HIV entry process and provide an overview of the design, antiviral potency, and methods of delivery of bAVPs. Additionally, we discuss the advantages and limitations of bAVPs for HIV prevention and treatment.