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1.
PLoS Pathog ; 17(7): e1009494, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34237110

RESUMO

The mammalian orthoreovirus double-stranded (ds) RNA-binding protein σ3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of σ3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of σ3 binding to dsRNA during viral infection is largely unknown. To identify functions of σ3 dsRNA-binding activity during reovirus infection, we engineered a panel of thirteen σ3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of σ3. Two recombinant viruses expressing these σ3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are formed at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not mediate formation of SGs at late times post-infection. These findings indicate that σ3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that σ3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice.


Assuntos
Miocardite/virologia , Proteínas de Ligação a RNA/metabolismo , Infecções por Reoviridae/metabolismo , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Células A549 , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Miocardite/metabolismo , eIF-2 Quinase/metabolismo
2.
Vaccines (Basel) ; 9(4)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916180

RESUMO

The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of vaccine candidates for this disease and other emerging diseases. Several novel methods for preparing vaccine candidates are currently undergoing clinical evaluation in response to the urgent need to prevent the spread of COVID-19. In many cases, these methods rely on new approaches for vaccine production and immune stimulation. We report on the use of a novel method (SolaVAX) for production of an inactivated vaccine candidate and the testing of that candidate in a hamster animal model for its ability to prevent infection upon challenge with SARS-CoV-2 virus. The studies employed in this work included an evaluation of the levels of neutralizing antibody produced post-vaccination, levels of specific antibody sub-types to RBD and spike protein that were generated, evaluation of viral shedding post-challenge, flow cytometric and single cell sequencing data on cellular fractions and histopathological evaluation of tissues post-challenge. The results from this preliminary evaluation provide insight into the immunological responses occurring as a result of vaccination with the proposed vaccine candidate and the impact that adjuvant formulations, specifically developed to promote Th1 type immune responses, have on vaccine efficacy and protection against infection following challenge with live SARS-CoV-2. This data may have utility in the development of effective vaccine candidates broadly. Furthermore, the results of this preliminary evaluation suggest that preparation of a whole virion vaccine for COVID-19 using this specific photochemical method may have potential utility in the preparation of one such vaccine candidate.

3.
Viruses ; 12(11)2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33228135

RESUMO

Serpentoviruses are an emerging group of nidoviruses known to cause respiratory disease in snakes and have been associated with disease in other non-avian reptile species (lizards and turtles). This study describes multiple episodes of respiratory disease-associated mortalities in a collection of juvenile veiled chameleons (Chamaeleo calyptratus). Histopathologic lesions included rhinitis and interstitial pneumonia with epithelial proliferation and abundant mucus. Metagenomic sequencing detected coinfection with two novel serpentoviruses and a novel orthoreovirus. Veiled chameleon serpentoviruses are most closely related to serpentoviruses identified in snakes, lizards, and turtles (approximately 40-50% nucleotide and amino acid identity of ORF1b). Veiled chameleon orthoreovirus is most closely related to reptilian orthoreoviruses identified in snakes (approximately 80-90% nucleotide and amino acid identity of the RNA-dependent RNA polymerase). A high prevalence of serpentovirus infection (>80%) was found in clinically healthy subadult and adult veiled chameleons, suggesting the potential for chronic subclinical carriers. Juvenile veiled chameleons typically exhibited a more rapid progression compared to subadults and adults, indicating a possible age association with morbidity and mortality. This is the first description of a serpentovirus infection in any chameleon species. A causal relationship between serpentovirus infection and respiratory disease in chameleons is suspected. The significance of orthoreovirus coinfection remains unknown.


Assuntos
Coinfecção/veterinária , Lagartos/virologia , Doenças Pulmonares Intersticiais/veterinária , Nidovirales/patogenicidade , Orthoreovirus/patogenicidade , Infecções por Reoviridae/veterinária , Animais , Animais de Zoológico/virologia , Coinfecção/virologia , Surtos de Doenças/veterinária , Feminino , Doenças Pulmonares Intersticiais/virologia , Masculino , Metagenômica , Nidovirales/genética , Orthoreovirus/genética , Prevalência
4.
J Virol ; 94(20)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32759315

RESUMO

Partitiviruses are segmented, multipartite double-stranded RNA (dsRNA) viruses that until recently were only known to infect fungi, plants, and protozoans. Metagenomic surveys have revealed that partitivirus-like sequences are also commonly associated with arthropods. One arthropod-associated partitivirus, galbut virus, is common in wild populations of Drosophila melanogaster To begin to understand the processes that underlie this virus's high global prevalence, we established colonies of wild-caught infected flies. Infection remained at stably high levels over 3 years, with between 63 and 100% of individual flies infected. Galbut virus infects fly cells and replicates in tissues throughout infected adults, including reproductive tissues and the gut epithelium. We detected no evidence of horizontal transmission via ingestion, but vertical transmission from either infected females or infected males was ∼100% efficient. Vertical transmission of a related partitivirus, verdadero virus, that we discovered in a laboratory colony of Aedes aegypti mosquitoes was similarly efficient. This suggests that efficient biparental vertical transmission may be a feature of at least a subset of insect-infecting partitiviruses. To study the impact of galbut virus infection free from the confounding effect of other viruses, we generated an inbred line of flies with galbut virus as the only detectable virus infection. We were able to transmit infection experimentally via microinjection of homogenate from these galbut-only flies. This sets the stage for experiments to understand the biological impact and possible utility of partitiviruses infecting model organisms and disease vectors.IMPORTANCE Galbut virus is a recently discovered partitivirus that is extraordinarily common in wild populations of the model organism Drosophila melanogaster Like for most viruses discovered through metagenomics, most of the basic biological questions about this virus remain unanswered. We found that galbut virus, along with a closely related partitivirus found in Aedes aegypti mosquitoes, is transmitted from infected females or males to offspring with ∼100% efficiency and can be maintained in laboratory colonies over years. This efficient transmission mechanism likely underlies the successful spread of these viruses through insect populations. We created Drosophila lines that contained galbut virus as the only virus infection and showed that these flies can be used as a source for experimental infections. This provides insight into how arthropod-infecting partitiviruses may be maintained in nature and sets the stage for exploration of their biology and potential utility.


Assuntos
Aedes/virologia , Vírus de RNA de Cadeia Dupla/metabolismo , Animais , Drosophila melanogaster , Feminino , Masculino
5.
Front Vet Sci ; 6: 338, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31632990

RESUMO

The aim of this study of serpentovirus infection in captive snakes was to assess the susceptibility of different types of snakes to infection and disease, to survey viral genetic diversity, and to evaluate management practices that may limit infection and disease. Antemortem oral swabs were collected from 639 snakes from 12 US collections, including 62 species, 28 genera, and 6 families: Pythonidae (N = 414 snakes; pythons were overrepresented in the sample population), Boidae (79), Colubridae (116), Lamprophiidae (4), Elapidae (12), and Viperidae (14). Infection was more common in pythons (38%; 95% CI: 33.1-42.4%), and in boas (10%; 95% CI: 5.2-18.7%) than in colubrids (0.9%, 95% CI: <0.01-4.7%); infection was not detected in other snake families (lamprophiids 0/4, 95% CI: 0-49%; elapids 0/12, 95% CI: 0-24.2%; and vipers 0/14, 95% CI: 0-21.5%), but more of these snakes need to be tested to confirm these findings. Clinical signs of respiratory disease were common in infected pythons (85 of 144). Respiratory signs were only observed in 1 of 8 infected boas and were absent in the single infected colubrid. Divergent serpentoviruses were detected in pythons, boas, and colubrids, suggesting that different serpentoviruses might vary in their ability to infect snakes of different families. Older snakes were more likely to be infected than younger snakes (p-value < 0.001) but males and females were equally likely to be infected (female prevalence: 23.4%, 95% CI 18.7-28.9%; male prevalence: 23.5%, 95% CI 18-30.1%; p-value = 0.144). Neither age (p-value = 0.32) nor sex (p-value = 0.06) was statistically associated with disease severity. Longitudinal sampling of pythons in a single collection over 28 months revealed serpentovirus infection is persistent, and viral clearance was not observed. In this collection, infection was associated with significantly increased rates of mortality (p-value = 0.001) with death of 75% of infected pythons and no uninfected pythons over this period. Offspring of infected parents were followed: vertical transmission either does not occur or occurs with a much lower efficiency than horizontal transmission. Overall, these findings confirm that serpentoviruses pose a significant threat to the health of captive python populations and can cause infection in boa and colubrid species.

6.
Wiley Interdiscip Rev RNA ; 10(5): e1536, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31034160

RESUMO

Numerous post-transcriptional RNA processes play a major role in regulating the quantity, quality and diversity of gene expression in the cell. These include RNA processing events such as capping, splicing, polyadenylation and modification, but also aspects such as RNA localization, decay, translation, and non-coding RNA-associated regulation. The interface between the transcripts of RNA viruses and the various RNA regulatory processes in the cell, therefore, has high potential to significantly impact virus gene expression, regulation, cytopathology and pathogenesis. Furthermore, understanding RNA biology from the perspective of an RNA virus can shed considerable light on the broad impact of these post-transcriptional processes in cell biology. Thus the goal of this article is to provide an overview of the richness of cellular RNA biology and how RNA viruses use, usurp and/or avoid the associated machinery to impact the outcome of infection. This article is categorized under: RNA in Disease and Development > RNA in Disease.


Assuntos
Vírus de RNA/metabolismo , RNA Viral/metabolismo , Vírus de RNA/genética , RNA Viral/genética
7.
Viruses ; 10(7)2018 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-30037148

RESUMO

Ixodes scapularis ticks harbor a variety of microorganisms, including eukaryotes, bacteria and viruses. Some of these can be transmitted to and cause disease in humans and other vertebrates. Others are not pathogenic, but may impact the ability of the tick to harbor and transmit pathogens. A growing number of studies have examined the influence of bacteria on tick vector competence but the influence of the tick virome remains less clear, despite a surge in the discovery of tick-associated viruses. In this study, we performed shotgun RNA sequencing on 112 individual adult I. scapularis collected in Wisconsin, USA. We characterized the abundance, prevalence and co-infection rates of viruses, bacteria and eukaryotic microorganisms. We identified pairs of tick-infecting microorganisms whose observed co-infection rates were higher or lower than would be expected, or whose RNA levels were positively correlated in co-infected ticks. Many of these co-occurrence and correlation relationships involved two bunyaviruses, South Bay virus and blacklegged tick phlebovirus-1. These viruses were also the most prevalent microorganisms in the ticks we sampled, and had the highest average RNA levels. Evidence of associations between microbes included a positive correlation between RNA levels of South Bay virus and Borrelia burgdorferi, the Lyme disease agent. These findings contribute to the rationale for experimental studies on the impact of viruses on tick biology and vector competence.


Assuntos
Coinfecção/microbiologia , Coinfecção/virologia , Ixodes/microbiologia , Ixodes/virologia , Doenças Transmitidas por Carrapatos/epidemiologia , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Borrelia burgdorferi/genética , Borrelia burgdorferi/isolamento & purificação , Coinfecção/epidemiologia , Eucariotos/genética , Eucariotos/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Ixodes/genética , Doença de Lyme , Microbiota/genética , Orthobunyavirus/genética , Orthobunyavirus/isolamento & purificação , Phlebovirus/genética , Phlebovirus/isolamento & purificação , Prevalência , Simbiose , Infestações por Carrapato/epidemiologia , Infestações por Carrapato/microbiologia , Infestações por Carrapato/virologia , Doenças Transmitidas por Carrapatos/microbiologia , Doenças Transmitidas por Carrapatos/virologia , Vírus/genética , Vírus/isolamento & purificação , Wisconsin/epidemiologia
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