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African swine fever virus (ASFV) is the causative agent of the highly lethal African swine fever disease that affects domestic pigs and wild boars. In spite of the rapid spread of the virus worldwide, there is no licensed vaccine available. The lack of a suitable cell line for ASFV propagation hinders the development of a safe and effective vaccine. For ASFV propagation, primary swine macrophages and monocytes have been widely studied. However, obtaining these cells can be time-consuming and expensive, making them unsuitable for mass vaccine production. The goal of this study was to validate the suitability of novel CA-CAS-01-A (CAS-01) cells, which was identified as a highly permissive cell clone for ASFV replication in the MA-104 parental cell line for live attenuated vaccine development. Through a screening experiment, maximum ASFV replication was observed in the CAS-01 cell compared to other sub-clones of MA-104 with 14.89 and log10 7.5 ± 0.15 Ct value and TCID50/ml value respectively. When CAS-01 cells are inoculated with ASFV, replication of ASFV was confirmed by Ct value for ASFV DNA, HAD50/ml assay, TCID50/ml assay, and cytopathic effects and hemadsoption were observed similar to those in primary porcine alveolar macrophages after 5th passage. Additionally, we demonstrated stable replication and adaptation of ASFV over the serial passage. These results suggest that CAS-01 cells will be a valuable and promising cell line for ASFV isolation, replication, and development of live attenuated vaccines.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Porcinos , Animales , Virus de la Fiebre Porcina Africana/genética , Fiebre Porcina Africana/prevención & control , Vacunas Atenuadas/genética , Proteínas Virales/genética , Sus scrofa , Desarrollo de Vacunas , Línea CelularRESUMEN
Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE: Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.
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Aptitud Genética , Hepacivirus , Hepatocitos , Interacciones Microbiota-Huesped , Inmunidad Innata , Mutación , Humanos , Células Cultivadas , Estrés del Retículo Endoplásmico , Aptitud Genética/genética , Aptitud Genética/inmunología , Hepacivirus/genética , Hepacivirus/crecimiento & desarrollo , Hepacivirus/inmunología , Hepacivirus/fisiología , Hepatitis C/inmunología , Hepatitis C/virología , Hepatocitos/inmunología , Hepatocitos/virología , Interacciones Microbiota-Huesped/inmunología , MicroARNs/metabolismo , Pase Seriado , Respuesta de Proteína Desplegada , Tropismo Viral , Virión/crecimiento & desarrollo , Virión/metabolismo , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants which underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establishing persistence.
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The relevance of tobamoviruses to crop production is increasing due to new emergences, which cannot be understood without knowledge of the tobamovirus host range and host specificity. Recent analyses of tobamovirus occurrence in different plant communities have shown unsuspectedly large host ranges. This was the case of the tobacco mild green mosaic virus (TMGMV), which previously was most associated with solanaceous hosts. We addressed two hypotheses concerning TMGMV host range evolution: (i) ecological fitting, rather than genome evolution, determines TMGMV host range, and (ii) isolates are adapted to the host of origin. We obtained TMGMV isolates from non-solanaceous hosts and we tested the capacity of genetically closely related TMGMV isolates from three host families to infect and multiply in 10 hosts of six families. All isolates systemically infected all hosts, with clear disease symptoms apparent only in solanaceous hosts. TMGMV multiplication depended on the assayed host but not on the isolate's host of origin, with all isolates accumulating to the highest levels in Nicotiana tabacum. Thus, results support that TMGMV isolates are adapted to hosts in the genus Nicotiana, consistent with a well-known old virus-host association. In addition, phenotypic plasticity allows Nicotiana-adapted TMGMV genotypes to infect a large range of hosts, as encountered according to plant community composition and transmission dynamics.
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Virus del Mosaico del Tabaco , Tobamovirus , ARN Viral/genética , Tobamovirus/genética , Nicotiana , Adaptación Fisiológica , Enfermedades de las PlantasRESUMEN
Plant viruses encounter a range of host defenses including non-host resistance (NHR), leading to the arrest of virus replication and movement in plants. Viruses have limited host ranges, and adaptation to a new host is an atypical phenomenon. The entire genotypes of plant species which are imperceptive to every single isolate of a genetically variable virus species are described as non-hosts. NHR is the non-specific resistance manifested by an innately immune non-host due to pre-existing and inducible defense responses, which cannot be evaded by yet-to-be adapted plant viruses. NHR-to-plant viruses are widespread, but the phenotypic variation is often not detectable within plant species. Therefore, molecular and genetic mechanisms of NHR need to be systematically studied to enable exploitation in crop protection. This article comprehensively describes the possible mechanisms of NHR against plant viruses. Also, the previous definition of NHR to plant viruses is insufficient, and the main aim of this article is to sensitize plant pathologists to the existence of NHR to plant viruses and to highlight the need for immediate and elaborate research in this area.
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Virus de Plantas , Plantas , Plantas/genética , Especificidad del Huésped , Aclimatación , Adaptación Fisiológica , Virus de Plantas/genéticaRESUMEN
Background and objectives: To understand how organisms evolve, it is fundamental to study how mutations emerge and establish. Here, we estimated the rate of mutation accumulation of SARS-CoV-2 in vitro and investigated the repeatability of its evolution when facing a new cell type but no immune or drug pressures. Methodology: We performed experimental evolution with two strains of SARS-CoV-2, one carrying the originally described spike protein (CoV-2-D) and another carrying the D614G mutation that has spread worldwide (CoV-2-G). After 15 passages in Vero cells and whole genome sequencing, we characterized the spectrum and rate of the emerging mutations and looked for evidences of selection across the genomes of both strains. Results: From the frequencies of the mutations accumulated, and excluding the genes with signals of selection, we estimate a spontaneous mutation rate of 1.3 × 10 -6 ± 0.2 × 10-6 per-base per-infection cycle (mean across both lineages of SARS-CoV-2 ± 2SEM). We further show that mutation accumulation is larger in the CoV-2-D lineage and heterogeneous along the genome, consistent with the action of positive selection on the spike protein, which accumulated five times more mutations than the corresponding genomic average. We also observe the emergence of mutators in the CoV-2-G background, likely linked to mutations in the RNA-dependent RNA polymerase and/or in the error-correcting exonuclease protein. Conclusions and implications: These results provide valuable information on how spontaneous mutations emerge in SARS-CoV-2 and on how selection can shape its genome toward adaptation to new environments. Lay Summary: Each time a virus replicates inside a cell, errors (mutations) occur. Here, via laboratory propagation in cells originally isolated from the kidney epithelium of African green monkeys, we estimated the rate at which the SARS-CoV-2 virus mutates-an important parameter for understanding how it can evolve within and across humans. We also confirm the potential of its Spike protein to adapt to a new environment and report the emergence of mutators-viral populations where mutations occur at a significantly faster rate.
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Understanding how animal influenza A viruses (IAVs) acquire airborne transmissibility in humans and ferrets is needed to prepare for and respond to pandemics. Here, we investigated in ferrets the replication and transmission of swine H1N1 isolates P4 and G15, whose majority population had decreased polymerase activity and poor hemagglutinin (HA) stability, respectively. For both isolates, a minor variant was selected and transmitted in ferrets. Polymerase-enhancing variant PA-S321 airborne-transmitted and propagated in one ferret. HA-stabilizing variant HA1-S210 was selected in all G15-inoculated ferrets and was transmitted by contact and airborne routes. With an efficient polymerase and a stable HA, the purified minor variant G15-HA1-S210 had earlier and higher peak titers in inoculated ferrets and was recovered at a higher frequency after airborne transmission than P4 and G15. Overall, HA stabilization played a more prominent role than polymerase enhancement in the replication and transmission of these viruses in ferrets. The results suggest pandemic risk-assessment studies may benefit from deep sequencing to identify minor variants with human-adapted traits. IMPORTANCE Diverse IAVs circulate in animals, yet few acquire the viral traits needed to start a human pandemic. A stabilized HA and mammalian-adapted polymerase have been shown to promote the adaptation of IAVs to humans and ferrets (the gold-standard model for IAV replication, pathogenicity, and transmissibility). Here, we used swine IAV isolates of the gamma lineage as a model to investigate the importance of HA stability and polymerase activity in promoting replication and transmission in ferrets. These are emerging viruses that bind to both α-2,6- and α-2,3-linked receptors. Using isolates containing mixed populations, a stabilized HA was selected within days in inoculated ferrets. An enhanced polymerase was also selected and propagated after airborne transmission to a ferret. Thus, HA stabilization was a stricter requirement, yet both traits promoted transmissibility. Knowing the viral traits needed for pandemic potential, and the relative importance of each, will help identify emerging viruses of greatest concern.
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Glicoproteínas Hemaglutininas del Virus de la Influenza , Subtipo H1N1 del Virus de la Influenza A , Infecciones por Orthomyxoviridae , Animales , Hurones , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/virología , Estabilidad Proteica , PorcinosRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing an unprecedented pandemic. Although vaccines and antivirals are limiting the spread, SARS-CoV-2 is still under selective pressure in human and animal populations, as demonstrated by the emergence of variants of concern. To better understand the driving forces leading to new subtypes of SARS-CoV-2, we infected an ex vivo cell model of the human upper respiratory tract with Alpha and Omicron BA.1 variants for one month. Although viral RNA was detected during the entire course of the infection, infectious virus production decreased over time. Sequencing analysis did not show any adaptation in the spike protein, suggesting a key role for the adaptive immune response or adaptation to other anatomical sites for the evolution of SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , Animales , Humanos , Antivirales , Nariz , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Tráquea , Evolución MolecularRESUMEN
The contribution of the HCPro factors expressed by several PVY isolates of different geographical origins (one from Scotland, one from Spain, and several from Tunisia) to differences in their virulence in Nicotiana benthamiana plants was investigated under two growing conditions: standard (st; 26 °C and current ambient levels of CO2), and climate change-associated (cc; 31 °C and elevated levels of CO2). In all cases, relative infection symptoms and viral titers were determined. The viral HCPro cistrons were also sequenced and amino-acid features of the encoded proteins were established, as well as phylogenetic distances. Additionally, the abilities of the HCPros of several isolates to suppress silencing were assessed under either growing condition. Overall, viral titers and infection symptoms decreased under cc vs. st conditions. However, within each growing condition, relative titers and symptoms were found to be isolate-specific, with titers and symptom severities not always correlating. Crucially, isolates expressing identical HCPros displayed different symptoms. In addition, all HCPro variants tested displayed comparable silencing suppression strengths. Therefore, HCPro alone could not be the main determinant of the relative differences in pathogenicity observed among the PVY isolates tested in this host, under the environments considered.
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Oncolytic virotherapy represents one of the most advanced strategies to treat otherwise untreatable types of cancer. Despite encouraging developments in recent years, the limited fraction of patients responding to therapy has demonstrated the need to search for new suitable viruses. Coxsackievirus B3 (CVB3) is a promising novel candidate with particularly valuable features. Its entry receptor, the coxsackievirus and adenovirus receptor (CAR), and heparan sulfate, which is used for cellular entry by some CVB3 variants, are highly expressed on various cancer types. Consequently, CVB3 has broad anti-tumor activity, as shown in various xenograft and syngeneic mouse tumor models. In addition to direct tumor cell killing the virus induces a strong immune response against the tumor, which contributes to a substantial increase in the efficiency of the treatment. The toxicity of oncolytic CVB3 in healthy tissues is variable and depends on the virus strain. It can be abrogated by genetic engineering the virus with target sites of microRNAs. In this review, we present an overview of the current status of the development of CVB3 as an oncolytic virus and outline which steps still need to be accomplished to develop CVB3 as a therapeutic agent for clinical use in cancer treatment.
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Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Virus Oncolíticos/fisiología , Animales , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Infecciones por Coxsackievirus/virología , Ingeniería Genética , Humanos , Ratones , MicroARNs , Receptores Virales/metabolismo , Internalización del VirusRESUMEN
Despite continuous and extensive efforts to control infectious bronchitis (IB) throughout the century, the disease continues to be one of the most economically relevant diseases affecting the poultry production worldwide. Since the early 1990s, numerous scientists have explicitly warned about the role of attenuated vaccines on IB virus (IBV) evolution and the detrimental consequences of their use to the poultry industry. Herein, we review evidence indicating that the use of live vaccines increases genetic/phenotypic diversity of IBV, enhances their fitness in the environment, and ultimately aggravates and perpetuates the problem for the poultry industry. The available evidence leads to the unequivocal conclusion that attenuated IBV vaccines should be replaced by vaccines using alternative technologies if IBV is to be controlled effectively.
Estudio recapitulativo- El control global de la bronquitis infecciosa requiere reemplazar las vacunas atenuadas por tecnologías alternativas. A pesar de los continuos y extensos esfuerzos para controlar el virus de la bronquitis infecciosa a lo largo del siglo, la bronquitis infecciosa sigue siendo una de las enfermedades de mayor relevancia económica que afecta a la producción avícola en todo el mundo. Desde principios de la década de los 1990s, numerosos científicos han advertido explícitamente sobre el papel de las vacunas atenuadas en la evolución del virus de la bronquitis infecciosa y las consecuencias perjudiciales de su uso para la industria avícola. En este artículo, se revisa la evidencia que indica que el uso de vacunas vivas aumenta la diversidad genética y fenotípica del virus de la bronquitis infecciosa, mejora su aptitud en el medio ambiente y en última instancia, agrava y perpetúa el problema para la industria avícola. La evidencia disponible lleva a la conclusión inequívoca de que las vacunas atenuadas contra este virus deben ser reemplazadas por vacunas que utilicen tecnologías alternativas si se quiere controlar eficazmente a este virus.
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Bronquitis , Infecciones por Coronavirus , Virus de la Bronquitis Infecciosa , Enfermedades de las Aves de Corral , Vacunas Virales , Animales , Bronquitis/veterinaria , Pollos , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/veterinaria , Virus de la Bronquitis Infecciosa/genética , Enfermedades de las Aves de Corral/prevención & control , Vacunas AtenuadasRESUMEN
Severe acute respiratory syndrome coronavirus 2 has caused a pandemic in humans. Farmed mink (Neovison vison) are also susceptible. In Denmark, this virus has spread rapidly among farmed mink, resulting in some respiratory disease. Full-length virus genome sequencing revealed novel virus variants in mink. These variants subsequently appeared within the local human community.
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COVID-19/transmisión , Transmisión de Enfermedad Infecciosa/veterinaria , Visón/virología , SARS-CoV-2/genética , Zoonosis Virales/transmisión , Animales , COVID-19/veterinaria , COVID-19/virología , Dinamarca/epidemiología , Granjas , Humanos , Zoonosis Virales/virologíaRESUMEN
Our current understanding of the natural evolution of RNA viruses comes largely from consensus level genetic analyses which ignore the diverse mutant swarms that comprise within-host viral populations. The breadth and composition of viral mutant swarms impact viral fitness and adaptation, and the capacity for swarm plasticity is likely to be particularly important for arthropod-borne viruses (arboviruses) that cycle between taxonomically divergent hosts. Despite this, characterization of the relationship between the selective pressures and genetic signatures of the mutant swarm and consensus sequences is lacking. To clarify this, we analyzed previously generated whole genome, deep-sequencing data from 548 West Nile virus samples isolated from avian tissues or mosquitoes in New York State from 1999-2018. Both consensus level (interhost) and minority level (intrahost) nucleotide and amino acid sequences were analyzed, and diversity at each position was calculated across the genome in order to assess the relationship between minority and consensus sequences for individual genes and hosts. Our results indicate that consensus sequences are an inept representation of the overall genetic diversity. Unique host and gene-specific signatures and selective pressures were identified. These data demonstrate that an accurate and comprehensive understanding of arbovirus evolution and adaptation within and between hosts requires consideration of minority genotypes.
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Variación Genética/genética , Interacciones Microbiota-Huesped/genética , Virus del Nilo Occidental/genética , Animales , Evolución Biológica , Aves/genética , Aves/virología , Culicidae/genética , Culicidae/virología , Evolución Molecular , Genoma Viral/genética , Genotipo , Caballos/genética , Caballos/virología , Mosquitos Vectores/genética , Mutación/genética , New York , Análisis de Secuencia de ADN/métodos , Secuenciación Completa del Genoma/métodosRESUMEN
Coxsackie B (CVB) viruses have been associated with type 1 diabetes. We have recently observed that CVB1 was linked to the initiation of the autoimmune process leading to type 1 diabetes in Finnish children. Viral persistency in the pancreas is currently considered as one possible mechanism. In the current study persistent infection was established in pancreatic ductal and beta cell lines (PANC-1 and 1.1B4) using four different CVB1 strains, including the prototype strain and three clinical isolates. We sequenced 5' untranslated region (UTR) and regions coding for structural and non-structural proteins and the second single open reading frame (ORF) protein of all persisting CVB1 strains using next generation sequencing to identify mutations that are common for all of these strains. One mutation, K257R in VP1, was found from all persisting CVB1 strains. The mutations were mainly accumulated in viral structural proteins, especially at BC, DE, EF loops and C-terminus of viral capsid protein 1 (VP1), the puff region of VP2, the knob region of VP3 and infection-enhancing epitope of VP4. This showed that the capsid region of the viruses sustains various changes during persistency some of which could be hallmark(s) of persistency.
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The time to the onset of AIDS symptoms in an HIV infected individual is known to correlate inversely with viremia and the level of immune activation. The correlation exists against the background of strong individual fluctuations demonstrating the existence of hidden variables depending on patient and virus parameters. At the moment, prognosis of the time to AIDS based on patient parameters is not possible. In addition, it is of paramount importance to understand the reason of progression to AIDS in untreated patients to be able to learn to control it by means other than anti-retroviral therapy. Here we develop a mechanistic mathematical model to predict the speed of progression to AIDS in individual untreated patients and patients treated with suboptimal therapy, based on a single-time measurement of several virological and immunological parameters. We show that the gradual increase in virus fitness during a chronic infection causes slow gradual depletion of CD4 T cells. Using the existing evolution models of HIV, we obtain general expressions predicting the time to the onset of AIDS symptoms in terms of the patient parameters, for low-viremia and high-viremia patients separately. We show that the evolution model of AIDS fits the existing data on virus-time correlations better than the alternative model of the deregulation of homeostatic response.
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Pandemic influenza A viruses can emerge from swine, an intermediate host that supports adaptation of human-preferred receptor-binding specificity by the hemagglutinin (HA) surface antigen. Other HA traits necessary for pandemic potential are poorly understood. For swine influenza viruses isolated in 2009-2016, gamma-clade viruses had less stable HA proteins (activation pH 5.5-5.9) than pandemic clade (pH 5.0-5.5). Gamma-clade viruses replicated to higher levels in mammalian cells than pandemic clade. In ferrets, a model for human adaptation, a relatively stable HA protein (pH 5.5-5.6) was necessary for efficient replication and airborne transmission. The overall airborne transmission frequency in ferrets for four isolates tested was 42%, and isolate G15 airborne transmitted 100% after selection of a variant with a stabilized HA. The results suggest swine influenza viruses containing both a stabilized HA and alpha-2,6 receptor binding in tandem pose greater pandemic risk. Increasing evidence supports adding HA stability to pre-pandemic risk assessment algorithms.
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Hurones , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H1N1 del Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/veterinaria , Animales , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Infecciones por Orthomyxoviridae/virologíaRESUMEN
Orthotospoviruses are plant-infecting members of the family Tospoviridae (order Bunyavirales), have a broad host range and are vectored by polyphagous thrips in a circulative-propagative manner. Because diverse hosts and vectors impose heterogeneous selection constraints on viral genomes, the evolutionary arms races between hosts and their pathogens might be manifested as selection for rapid changes in key genes. These observations suggest that orthotospoviruses contain key genetic components that rapidly mutate to mediate host adaptation and vector transmission. Using complete genome sequences, we profiled genomic variation in orthotospoviruses. Results show that the three genomic segments contain hypervariable areas at homologous locations across species. Remarkably, the highest nucleotide variation mapped to the intergenic region of RNA segments S and M, which fold into a hairpin. Secondary structure analyses showed that the hairpin is a dynamic structure with multiple functional shapes formed by stems and loops, contains sites under positive selection and covariable sites. Accumulation and tolerance of mutations in the intergenic region is a general feature of orthotospoviruses and might mediate adaptation to host plants and insect vectors.
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Live oral rotavirus vaccines have been developed by serial passaging in cell culture and found to be safe in infants. However, mechanisms for the adaptation and attenuation of rotavirus vaccines are not fully understood. We prepared a human rotavirus vaccine strain, CDC-9 (G1P[8]), which when grown in MA104 cells to passage 11 or 12 (P11/P12) had no nucleotide or amino acid sequence changes from the original virus in stool. Upon adaptation and passages in Vero cells, the strain underwent five amino acid changes at P28 and one additional change at P44/P45 in the VP4 gene. We performed virologic, immunological, and pathogenic characterization of wild-type CDC-9 virus at P11/P12 and its two mutants at P28 or P44/P45 using in vitro and in vivo model systems. We found that mutants CDC-9 P28 and P44 induced upregulated expression of immunomodulatory cytokines. On the other hand, the two mutant viruses induced lower STAT1 phosphorylation and grew to 2-log-higher titers than wild-type virus in human Caco-2 cells and simian Vero cells. In neonatal rats, CDC-9 P45 showed reduced rotavirus shedding in fecal specimens and did not induce diarrhea compared to wild-type virus and modulated cytokine responses comparably to Rotarix infection. These findings indicate that mutant CDC-9 is attenuated and safe. Our study is the first to provide insight into the possible mechanisms of human rotavirus adaptation and attenuation and supports ongoing efforts to develop CDC-9 as a new generation of rotavirus vaccine for live oral or parenteral administration.IMPORTANCE Mechanisms for in vitro adaptation and in vivo attenuation of human rotavirus vaccines are not known. The present study is the first to comprehensively compare the in vitro growth characteristics, virulence, and host response of a wild-type and an attenuated human rotavirus strain, CDC-9, in Caco-2 cells and neonatal rats. Our study identifies critical sequence changes in the genome that render human rotavirus adapted to growth to high levels in Vero cells and attenuated and safe in neonatal rats; thus, the study supports clinical development of CDC-9 for oral or parenteral vaccination in children.
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Proteínas de la Cápside/metabolismo , Mutación Missense , Vacunas contra Rotavirus/metabolismo , Rotavirus/crecimiento & desarrollo , Sustitución de Aminoácidos , Animales , Células CACO-2 , Proteínas de la Cápside/genética , Chlorocebus aethiops , Humanos , Rotavirus/genética , Vacunas contra Rotavirus/genética , Vacunas Atenuadas/genética , Vacunas Atenuadas/metabolismo , Células VeroRESUMEN
Although epigenetic factors may influence the expression of defense genes in plants, their role in antiviral responses and the impact of viral adaptation and evolution in shaping these interactions are still poorly explored. We used two isolates of turnip mosaic potyvirus with varying degrees of adaptation to Arabidopsis thaliana to address these issues. One of the isolates was experimentally evolved in the plant and presented increased load and virulence relative to the ancestral isolate. The magnitude of the transcriptomic responses was larger for the evolved isolate and indicated a role of innate immunity systems triggered by molecular patterns and effectors in the infection process. Several transposable elements located in different chromatin contexts and epigenetic-related genes were also affected. Correspondingly, mutant plants having loss or gain of repressive marks were, respectively, more tolerant and susceptible to turnip mosaic potyvirus, with a more efficient response against the ancestral isolate. In wild-type plants, both isolates induced similar levels of cytosine methylation changes, including in and around transposable elements and stress-related genes. Results collectively suggested that apart from RNA silencing and basal immunity systems, DNA methylation and histone modification pathways may also be required for mounting proper antiviral defenses and that the effectiveness of this type of regulation strongly depends on the degree of viral adaptation to the host.
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Arabidopsis/virología , Epigénesis Genética , Aptitud Genética , Interacciones Huésped-Patógeno/inmunología , Potyvirus/fisiología , Adaptación Biológica , Arabidopsis/inmunología , Arabidopsis/metabolismo , Evolución Biológica , Metilación de ADN , TranscriptomaRESUMEN
Orthohantaviruses are re-emerging rodent-borne pathogens distributed all over the world. Here, we report the isolation of a Puumala orthohantavirus (PUUV) strain from bank voles caught in a highly endemic region around the city Osnabrück, north-west Germany. Coding and non-coding sequences of all three segments (S, M, and L) were determined from original lung tissue, after isolation and after additional passaging in VeroE6 cells and a bank vole-derived kidney cell line. Different single amino acid substitutions were observed in the RNA-dependent RNA polymerase (RdRP) of the two stable PUUV isolates. The PUUV strain from VeroE6 cells showed a lower titer when propagated on bank vole cells compared to VeroE6 cells. Additionally, glycoprotein precursor (GPC)-derived virus-like particles of a German PUUV sequence allowed the generation of monoclonal antibodies that allowed the reliable detection of the isolated PUUV strain in the immunofluorescence assay. In conclusion, this is the first isolation of a PUUV strain from Central Europe and the generation of glycoprotein-specific monoclonal antibodies for this PUUV isolate. The obtained virus isolate and GPC-specific antibodies are instrumental tools for future reservoir host studies.