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1.
Viruses ; 14(3)2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35336935

RESUMO

Modified vaccinia Ankara (MVA) is a promising vaccine vector due to its highly attenuated phenotype and good immunogenicity. However, obtaining a new recombinant MVA remains a tedious and laborious procedure involving many rounds of plaque purification. Recombinant MVA generation can be greatly improved and facilitated by different selection techniques. Here, we describe a comparison between techniques based on K1L, F13L and D4R genes.


Assuntos
Genes Virais , Vírus Vaccinia , Linhagem Celular , Vetores Genéticos/genética , Vírus Vaccinia/genética
2.
Methods Mol Biol ; 2408: 23-35, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35325414

RESUMO

RNA interference (RNAi) is an evolutionarily conserved gene silencing mechanism in eukaryotes including fungi, plants, and animals. In plants, gene silencing regulates gene expression, provides genome stability, and protect against invading viruses. During plant virus interaction, viral genome derived siRNAs (vsiRNA) are produced to mediate gene silencing of viral genes to prevent virus multiplication. After the discovery of RNAi phenomenon in eukaryotes, it is used as a powerful tool to engineer plant viral disease resistance against both RNA and DNA viruses. Despite several successful reports on employing RNA silencing methods to engineer plant for viral disease resistance, only a few of them have reached the commercial stage owing to lack of complete protection against the intended virus. Based on the knowledge accumulated over the years on genetic engineering for viral disease resistance, there is scope for effective viral disease control through careful design of RNAi gene construct. The selection of target viral gene(s) for developing the hairpin RNAi (hp-RNAi) construct is very critical for effective protection against the viral disease. Different approaches and bioinformatics tools which can be employed for effective target selection are discussed. The selection of suitable target regions for RNAi vector construction can help to achieve a high level of transgenic virus resistance.


Assuntos
Resistência à Doença , Vírus de Plantas , Animais , Resistência à Doença/genética , Inativação Gênica , Genes Virais , Vírus de Plantas/genética , Interferência de RNA
3.
Microbiol Spectr ; 10(1): e0068121, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35170989

RESUMO

The N501Y amino acid mutation caused by a single point substitution A23063T in the spike gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possessed by three variants of concern (VOCs), B.1.1.7, B.1.351, and P.1. A rapid screening tool using this mutation is important for surveillance during the coronavirus disease 2019 (COVID-19) pandemic. We developed and validated a single nucleotide polymorphism real-time reverse transcription PCR assay using allelic discrimination of the spike gene N501Y mutation to screen for potential variants of concern and differentiate them from SARS-CoV-2 lineages without the N501Y mutation. A total of 160 clinical specimens positive for SARS-CoV-2 were characterized as mutant (N501Y) or N501 wild type by Sanger sequencing and were subsequently tested with the N501Y single nucleotide polymorphism real-time reverse transcriptase PCR assay. Our assay, compared to Sanger sequencing for single nucleotide polymorphism detection, demonstrated positive percent agreement of 100% for all 57 specimens displaying the N501Y mutation, which were confirmed by Sanger sequencing to be typed as A23063T, including one specimen with mixed signal for wild type and mutant. Negative percent agreement was 100% in all 103 specimens typed as N501 wild type, with A23063 identified as wild type by Sanger sequencing. The identification of circulating SARS-CoV-2 lineages carrying an N501Y mutation is critical for surveillance purposes. Current identification methods rely primarily on Sanger sequencing or whole-genome sequencing, which are time consuming, labor intensive, and costly. The assay described herein is an efficient tool for high-volume specimen screening for SARS-CoV-2 VOCs and for selecting specimens for confirmatory Sanger or whole-genome sequencing. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, several variants of concern (VOCs) have been detected, for example, B.1.1.7, B.1.351, P.1, and B.1.617.2. The VOCs pose a threat to public health efforts to control the spread of the virus. As such, surveillance and monitoring of these VOCs is of the utmost importance. Our real-time RT-PCR assay helps with surveillance by providing an easy method to quickly survey SARS-CoV-2 specimens for VOCs carrying the N501Y single nucleotide polymorphism (SNP). Samples that test positive for the N501Y mutation in the spike gene with our assay can be sequenced to identify the lineage. Thus, our assay helps to focus surveillance efforts and decrease turnaround times.


Assuntos
COVID-19/diagnóstico , Mutação de Sentido Incorreto , Mutação Puntual , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Alelos , Substituição de Aminoácidos , COVID-19/epidemiologia , COVID-19/virologia , Genes Virais , Humanos , Programas de Rastreamento , Ontário/epidemiologia , Polimorfismo de Nucleotídeo Único , Vigilância da População , Prevalência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
4.
Viruses ; 14(2)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35215990

RESUMO

Sequences derived from a novel toursvirus were identified from pooled genomic short read data from U.S. populations of southern corn rootworm (SCR, Diabrotica undecimpunctata howardi Barber) and northern corn rootworm (NCR, Diabrotica barberi Smith & Lawrence). Most viral sequences were identified from the SCR genomic dataset. As proteins encoded by toursvirus sequences from SCR and NCR were almost identical, the contig sets from SCR and NCR were combined to generate 26 contigs. A total of 108,176 bp were assembled from these contigs, with 120 putative toursviral ORFs identified indicating that most of the viral genome had been recovered. These ORFs included all 40 genes that are common to members of the Ascoviridae. Two genes typically present in Ascoviridae (ATP binding cassette transport system permeases and Baculovirus repeated open reading frame), were not detected. There was evidence for transposon insertion in viral sequences at different sites in the two host species. Phylogenetic analyses based on a concatenated set of 45 translated protein sequences clustered toursviruses into a distinct clade. Based on the combined evidence, we propose taxonomic separation of toursviruses from Ascoviridae.


Assuntos
Ascoviridae/genética , Besouros/virologia , Animais , Ascoviridae/classificação , Besouros/classificação , DNA Viral/genética , Feminino , Genes Virais , Genoma Viral/genética , Genômica , Masculino , Fases de Leitura Aberta , Filogenia
5.
Front Immunol ; 13: 801915, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35211117

RESUMO

Due to the fast global spreading of the Severe Acute Respiratory Syndrome Coronavirus - 2 (SARS-CoV-2), prevention and treatment options are direly needed in order to control infection-related morbidity, mortality, and economic losses. Although drug and inactivated and attenuated virus vaccine development can require significant amounts of time and resources, DNA and RNA vaccines offer a quick, simple, and cheap treatment alternative, even when produced on a large scale. The spike protein, which has been shown as the most antigenic SARS-CoV-2 protein, has been widely selected as the target of choice for DNA/RNA vaccines. Vaccination campaigns have reported high vaccination rates and protection, but numerous unintended effects, ranging from muscle pain to death, have led to concerns about the safety of RNA/DNA vaccines. In parallel to these studies, several open reading frames (ORFs) have been found to be overlapping SARS-CoV-2 accessory genes, two of which, ORF2b and ORF-Sh, overlap the spike protein sequence. Thus, the presence of these, and potentially other ORFs on SARS-CoV-2 DNA/RNA vaccines, could lead to the translation of undesired proteins during vaccination. Herein, we discuss the translation of overlapping genes in connection with DNA/RNA vaccines. Two mRNA vaccine spike protein sequences, which have been made publicly-available, were compared to the wild-type sequence in order to uncover possible differences in putative overlapping ORFs. Notably, the Moderna mRNA-1273 vaccine sequence is predicted to contain no frameshifted ORFs on the positive sense strand, which highlights the utility of codon optimization in DNA/RNA vaccine design to remove undesired overlapping ORFs. Since little information is available on ORF2b or ORF-Sh, we use structural bioinformatics techniques to investigate the structure-function relationship of these proteins. The presence of putative ORFs on DNA/RNA vaccine candidates implies that overlapping genes may contribute to the translation of smaller peptides, potentially leading to unintended clinical outcomes, and that the protein-coding potential of DNA/RNA vaccines should be rigorously examined prior to administration.


Assuntos
Homologia de Genes , Genes Virais , Vacinas de DNA/genética , /genética , Vacinas contra COVID-19/efeitos adversos , Vacinas contra COVID-19/genética , Códon , Humanos , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Biossíntese de Proteínas , Domínios Proteicos , RNA Mensageiro , Glicoproteína da Espícula de Coronavírus/genética , Vacinas de DNA/efeitos adversos , /efeitos adversos
6.
ACS Synth Biol ; 11(2): 522-527, 2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35176864

RESUMO

The ability to construct, synthesize, and edit genes and genomes at scale and with speed enables, in synergy with other tools of engineering biology, breakthrough applications with far-reaching implications for society. As SARS-CoV-2 spread around the world in early spring of 2020, researchers rapidly mobilized, using these tools in the development of diagnostics, therapeutics, and vaccines for COVID-19. The sharing of knowledge was crucial to making rapid progress. Several publications described the use of reverse genetics for the de novo construction of SARS-CoV-2 in the laboratory, one in the form of a protocol. Given the demonstrable harm caused by the virus, the unequal distribution of mitigating vaccines and therapeutics, their unknown efficacy against variants, and the interest in this research by laboratories unaccustomed to working with highly transmissible pandemic pathogens, there are risks associated with such publications, particularly as protocols. We describe considerations and offer suggestions for enhancing security in the publication of synthetic biology research and techniques. We recommend: (1) that protocol manuscripts for the de novo synthesis of certain pathogenic viruses undergo a mandatory safety and security review; (2) that if published, such papers include descriptions of the discussions or review processes that occurred regarding security considerations in the main text; and (3) the development of a governance framework for the inclusion of basic security screening during the publication process of engineering biology/synthetic biology manuscripts to build and support a safe and secure research enterprise that is able to maximize its positive impacts and minimize any negative outcomes.


Assuntos
Bioengenharia , Editoração , Medidas de Segurança/organização & administração , Genes Virais , SARS-CoV-2/genética , Biologia Sintética
7.
PLoS One ; 17(2): e0264008, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35171960

RESUMO

The C29197T mutation is one of 4 point mutations known to cause N-gene target failure (NGTF) in the Xpert Xpress SARS-CoV-2 and Xpert Omni SARS-CoV-2 assays from Cepheid (Sunnyvale, CA). We describe a high local prevalence in January of 8.5% (CI 4.9-14.2%) for the C29197T mutation, which was over 3-fold higher than the prevalence estimated statewide in California during the same time frame, 2.5% (CI 2.1-2.8%). Using phylogenetic analysis, we discovered that this increase in prevalence was due, at least in part, to a disproportionately large infection cluster of unknown origin. This study emphasizes the importance of sequencing at the local jurisdictional level and demonstrates the impact that regional variation can have when assessing risk due to point mutations that impact clinical test performance. It also reinforces the need for diligent reporting of abnormal test results by clinical laboratories, especially during Emergency Use Authorization (EUA) periods, as additional information is gathered about the target organism and the performance of EUA-authorized tests over time.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Genes Virais , Humanos , Técnicas de Diagnóstico Molecular/métodos , Mutação , Filogenia , Prevalência , SARS-CoV-2/genética , Sensibilidade e Especificidade
9.
PLoS One ; 17(1): e0262170, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35051202

RESUMO

The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/epidemiologia , Genes Virais , Pandemias/prevenção & controle , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Análise de Sequência de RNA/métodos , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Bases , Brasil/epidemiologia , COVID-19/virologia , Testes Diagnósticos de Rotina/métodos , Eletroforese em Gel de Ágar/métodos , Monitoramento Epidemiológico , Humanos , Mutação , RNA Viral/genética , RNA Viral/isolamento & purificação
10.
PLoS One ; 17(1): e0262656, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35051208

RESUMO

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Testes Diagnósticos de Rotina/métodos , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Carga Viral/métodos , COVID-19/epidemiologia , COVID-19/virologia , Genes Virais , Alemanha/epidemiologia , Humanos , Reprodutibilidade dos Testes
11.
Viruses ; 14(1)2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-35062304

RESUMO

Viruses are a possible cause for Sjögren's syndrome (SS) as an environmental factor related to SS onset, which exhibits exocrine gland dysfunction and the emergence of autoantibodies. Although retroviruses may exhibit lymphocytic infiltration into exocrine glands, human T-cell leukemia virus type 1 (HTLV-1) has been postulated to be a causative agent for SS. Transgenic mice with HTLV-1 genes showed sialadenitis resembling SS, but their phenotypic symptoms differed based on the adopted region of HTLV-1 genes. The dominance of tax gene differed in labial salivary glands (LSGs) of SS patients with HTLV 1-associated myelopathy (HAM) and adult T-cell leukemia. Although HTLV-1 was transmitted to salivary gland epithelial cells (SGECs) by a biofilm-like structure, no viral synapse formation was observed. After infection to SGECs derived from SS patients, adhesion molecules and migration factors were time-dependently released from infected SGECs. The frequency of the appearance of autoantibodies including anti-Ro/SS-A, La/SS-B antibodies in SS patients complicated with HAM is unknown; the observation of less frequent ectopic germinal center formation in HTLV-1-seropositive SS patients was a breakthrough. In addition, HTLV-1 infected cells inhibited B-lymphocyte activating factor or C-X-C motif chemokine 13 through direct contact with established follicular dendritic cell-like cells. These findings show that HTLV-1 is directly involved in the pathogenesis of SS.


Assuntos
Infecções por HTLV-I , Síndrome de Sjogren/virologia , Animais , Autoanticorpos/biossíntese , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Genes Virais , Infecções por HTLV-I/complicações , Infecções por HTLV-I/epidemiologia , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Linfócitos/virologia , Camundongos , Camundongos Transgênicos , Paraparesia Espástica Tropical/complicações , Paraparesia Espástica Tropical/epidemiologia , Paraparesia Espástica Tropical/imunologia , Paraparesia Espástica Tropical/virologia , Fenótipo , Ratos , Proteínas dos Retroviridae/genética , Proteínas dos Retroviridae/metabolismo , Glândulas Salivares/citologia , Glândulas Salivares/metabolismo , Glândulas Salivares/virologia , Síndrome de Sjogren/epidemiologia , Síndrome de Sjogren/imunologia
12.
PLoS Pathog ; 18(1): e1010236, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35041709

RESUMO

While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel, enabling the pooled screening of entire viral genomes. Here, we applied this approach to Kaposi's sarcoma-associated herpesvirus (KSHV) by designing a sgRNA library containing all possible ~22,000 guides targeting the 154 kilobase viral genome, corresponding to one cut site approximately every 8 base pairs. We used the library to profile viral sequences involved in transcriptional activation of late genes, whose regulation involves several well characterized features including dependence on viral DNA replication and a known set of viral transcriptional activators. Upon phenotyping all possible Cas9-targeted viruses for transcription of KSHV late genes we recovered these established regulators and identified a new required factor (ORF46), highlighting the utility of the screening pipeline. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we identify the DNA binding but not catalytic domain of ORF46 to be required for viral DNA replication and thus late gene expression. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.


Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Genes Virais/genética , Herpesvirus Humano 8/genética , Sistemas CRISPR-Cas , Células HEK293 , Humanos
13.
Biochim Biophys Acta Mol Basis Dis ; 1868(2): 166291, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34662705

RESUMO

OBJECTIVES: To investigate in silico the presence of nucleotide sequence complementarity between the RNA genome of Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) and human non-coding (nc)RNA genes. METHODS: The FASTA sequence (NC_045512.2) of each of the 11 SARS-CoV-2 isolate Wuhan-Hu-1 genes was retrieved from NCBI.nlm.nih.gov/gene and the Ensembl.org library interrogated for any base-pair match with human ncRNA genes. SARS-CoV-2 gene-matched human ncRNAs were screened for functional activity using bioinformatic analysis. Finally, associations between identified ncRNAs and human diseases were searched in GWAS databases. RESULTS: A total of 252 matches were found between the nucleotide sequence of SARS-CoV-2 genes and human ncRNAs. With the exception of two small nuclear RNAs, all of them were long non-coding (lnc)RNAs expressed mainly in testis and central nervous system under physiological conditions. The percentage of alignment ranged from 91.30% to 100% with a mean nucleotide alignment length of 17.5 ± 2.4. Thirty-three (13.09%) of them contained predicted R-loop forming sequences, but none of these intersected the complementary sequences of SARS-CoV-2. However, in 31 cases matches fell on ncRNA regulatory sites, whose adjacent coding genes are mostly involved in cancer, immunological and neurological pathways. Similarly, several polymorphic variants of detected non-coding genes have been associated with neuropsychiatric and proliferative disorders. CONCLUSION: This pivotal in silico study shows that SARS-CoV-2 genes have Watson-Crick nucleotide complementarity to human ncRNA sequences, potentially disrupting ncRNA epigenetic control of target genes. It remains to be elucidated whether this could result in the development of human disease in the long term.


Assuntos
COVID-19/genética , COVID-19/virologia , RNA não Traduzido/genética , SARS-CoV-2/genética , Sequência de Bases , Epigênese Genética , Genes Virais , Humanos , Neoplasias/genética , RNA Longo não Codificante/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
14.
J Control Release ; 341: 44-50, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34785314

RESUMO

To meet the present and future challenges in achieving therapeutic in vivo gene delivery using adeno-associated virus (AAV), new innovations are required that integrate knowledge from disciplines ranging from biomaterials science, drug delivery, immunobiology, to tissue engineering. One of the foremost challenges remaining is in addressing pre-existing and therapy induced immune responses to AAV which significantly limit its therapeutic effect. In addition, functional correction of diseased tissues will depend on the ability of AAVs to retain activity after local or systemic administration and broadly distribute in target tissues. In this contribution to the Orations - New Horizons of the Journal of Controlled Release, I will introduce new concepts and potential strategies pursued by our lab and others to better understand and overcome these hurdles to effective AAV gene therapy. These multi-disciplinary approaches may open the door to the creation of precision gene therapies to treat heavily burdensome and often deadly diseases.


Assuntos
Dependovirus , Vetores Genéticos , Dependovirus/genética , Técnicas de Transferência de Genes , Genes Virais , Terapia Genética , Vetores Genéticos/genética
15.
Hum Cell ; 35(1): 408-417, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34817797

RESUMO

Ex vivo manufactured red blood cells (RBC) generated from immortalized erythroid cell lines which can continuously grow are expected to become a significant alternative in future transfusion therapies. The ectopic expression of human papilloma virus (HPV) E6/E7 gene has successfully been employed to establish these cell lines. To induce differentiation and maturation of the immortalized cell lines, terminating the HPV-E6/E7 expression through a gene induction system has been believed to be essential. Here, we report that erythroid cell lines established from human bone marrow using simple expression of HPV-E6/E7 are capable of normal erythroid differentiation, without turning gene expression off. Through simply changing cell culture conditions, a newly established cell line, Erythroid Line from Lund University (ELLU), is able to differentiate toward mature cells, including enucleated reticulocytes. ELLU is heterogeneous and, unexpectedly, clones expressing adult hemoglobin rapidly differentiate and produce fragile cells. Upon differentiation, other ELLU clones shift from fetal to adult hemoglobin expression, giving rise to more mature cells. Our findings propose that it is not necessary to employ gene induction systems to establish immortalized erythroid cell lines sustaining differentiation potential and describe novel cellular characteristics for desired functionally competent clones.


Assuntos
Diferenciação Celular , Células Eritroides , Expressão Gênica , Alphapapillomavirus/genética , Células da Medula Óssea , Linhagem Celular , Células Clonais , Genes Virais , Vetores Genéticos , Hemoglobinas , Humanos , Reticulócitos
16.
J Med Virol ; 94(2): 521-530, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34761827

RESUMO

Measles is one of the most infectious diseases of humans. It is caused by the measles virus (MeV) and can lead to serious illness, lifelong complications, and even death. Whole-genome sequencing (WGS) is now available to study molecular epidemiology and identify MeV transmission pathways. In the present study, WGS of 23 MeV strains of genotype H1, collected in Mainland China between 2006 and 2018, were generated and compared to 31 WGSs from the public domain to analyze genomic characteristics, evolutionary rates and date of emergence of H1 genotype. The noncoding region between M and F protein genes (M/F NCR) was the most variable region throughout the genome. Although the nucleotide substitution rate of H1 WGS was around 0.75 × 10-3 substitution per site per year, the M/F NCR had an evolutionary rate three times higher, with 2.44 × 10-3 substitution per site per year. Phylogenetic analysis identified three distinct genetic groups. The Time of the Most Recent Common Ancestor (TMRCA) of H1 genotype was estimated at approximately 1988, while the first genetic group appeared around 1995 followed by two other genetic groups in 1999-2002. Bayesian skyline plot showed that the genetic diversity of the H1 genotype remained stable even though the number of MeV cases decreased 50 times between 2014 (52 628) and 2020 (993). The current coronavirus disease 2019 (COVID-19) pandemic might have some effect on the measles epidemic and further studies will be necessary to assess the genetic diversity of the H1 genotype in a post-COVID area.


Assuntos
Evolução Molecular , Genoma Viral/genética , Vírus do Sarampo/genética , China/epidemiologia , Genes Virais/genética , Variação Genética , Genômica , Genótipo , Humanos , Sarampo/epidemiologia , Sarampo/virologia , Vírus do Sarampo/classificação , Filogenia , RNA Viral/genética
17.
J Virol ; 96(4): e0169321, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-34908446

RESUMO

Epstein-Barr virus (EBV) infection is associated with multiple malignancies, including pulmonary lymphoepithelioma-like carcinoma (pLELC), a particular subtype of primary lung cancer. However, the genomic characteristics of EBV related to pLELC remain unclear. Here, we obtained the whole-genome data set of EBV isolated from 78 pLELC patients and 37 healthy controls using EBV-captured sequencing. Compared with the reference genome (NC_007605), a total of 3,995 variations were detected across pLELC-derived EBV sequences, with the mutational hot spots located in latent genes. Combined with 180 published EBV sequences derived from healthy people in Southern China, we performed a genome-wide association study and identified 32 variations significantly related to pLELC (P < 2.56 × 10-05, Bonferroni correction), with the top signal of single nucleotide polymorphism (SNP) coordinate T7327C (OR = 1.22, P = 2.39 × 10-15) locating in the origin of plasmid replication (OriP). The results of population structure analysis of EBV isolates in East Asian showed the EBV strains derived from pLELC were more similar to those from nasopharyngeal carcinoma (NPC) than other EBV-associated diseases. In addition, typical latency type-II infection were recognized for EBV of pLELC at both transcription and methylation levels. Taken together, we defined the global view of EBV genomic profiles in pLELC patients for the first time, providing new insights to deepening our understanding of this rare EBV-associated primary lung carcinoma. IMPORTANCE Pulmonary lymphoepithelioma-like carcinoma (pLELC) is a rare, distinctive subtype of primary lung cancer closely associated with Epstein-Barr virus (EBV) infection. Here, we gave the first overview of pLELC-derived EBV at the level of genome, methylation and transcription. We obtained the EBV sequences data set from 78 primary pLELC patients, and revealed the sequences diversity across EBV genome and detected variability in known immune epitopes. Genome-wide association analysis combining 217 healthy controls identifies significant variations related to the risk of pLELC. Meanwhile, we characterized the integration landscapes of EBV at the genome-wide level. These results provided new insight for understanding EBV's role in pLELC tumorigenesis.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/virologia , Infecções por Vírus Epstein-Barr/virologia , Genoma Viral/genética , Herpesvirus Humano 4/genética , Neoplasias Pulmonares/virologia , China , Metilação de DNA , Epitopos de Linfócito T/genética , Genes Virais/genética , Variação Genética , Estudo de Associação Genômica Ampla , Herpesvirus Humano 4/isolamento & purificação , Humanos , Integração Viral , Latência Viral/genética
18.
Viruses ; 13(12)2021 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-34960652

RESUMO

Murine hepatitis virus strain A59 (MHV-A59) was shown to induce pyroptosis, apoptosis, and necroptosis of infected cells, especially in the murine macrophages. However, whether ferroptosis, a recently identified form of lytic cell death, was involved in the pathogenicity of MHV-A59 is unknown. We utilized murine macrophages and a C57BL/6 mice intranasal infection model to address this. In primary macrophages, the ferroptosis inhibitor inhibited viral propagation, inflammatory cytokines released, and cell syncytia formed after MHV-A59 infection. In the mouse model, we found that in vivo administration of liproxstatin-1 ameliorated lung inflammation and tissue injuries caused by MHV-A59 infection. To find how MHV-A59 infection influenced the expression of ferroptosis-related genes, we performed RNA-seq in primary macrophages and found that MHV-A59 infection upregulates the expression of the acyl-CoA synthetase long-chain family member 1 (ACSL1), a novel ferroptosis inducer. Using ferroptosis inhibitors and a TLR4 inhibitor, we showed that MHV-A59 resulted in the NF-kB-dependent, TLR4-independent ACSL1 upregulation. Accordingly, ACSL1 inhibitor Triacsin C suppressed MHV-A59-infection-induced syncytia formation and viral propagation in primary macrophages. Collectively, our study indicates that ferroptosis inhibition protects hosts from MHV-A59 infection. Targeting ferroptosis may serve as a potential treatment approach for dealing with hyper-inflammation induced by coronavirus infection.


Assuntos
Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/metabolismo , Infecções por Coronavirus/terapia , Ferroptose , Animais , Coenzima A Ligases/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Genes Virais , Lesão Pulmonar/patologia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Hepatite Murina , Quinoxalinas , Células RAW 264.7 , Compostos de Espiro , Receptor 4 Toll-Like , Replicação Viral/genética
19.
Sci Rep ; 11(1): 24394, 2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34937862

RESUMO

Staphylococcus aureus can be a harmless coloniser, but it can also cause severe infections in humans, livestock and wildlife. Regarding the latter, only few studies have been performed and knowledge on virulence factors is insufficient. The aim of the present study was to study S. aureus isolates from deceased wild beavers (Castor fiber). Seventeen isolates from eleven beavers, found in Germany and Austria, were investigated. Antimicrobial and biocide susceptibility tests were performed. Isolates were characterised using S. aureus-specific DNA microarrays, spa typing and whole-genome sequencing. From two isolates, prophages were induced by mitomycin C and studied by transmission electron microscopy. Four isolates belonged to clonal complex (CC) 8, CC12, and CC398. Twelve isolates belonged to CC1956 and one isolate was CC49. The CC49 and CC1956 isolates carried distinct lukF/S genes related to the Panton-Valentine leukocidin (PVL) from human isolates of S. aureus. These genes were located on related, but not identical, Siphovirus prophages. The beavers, from which those isolates originated, suffered from abscesses, purulent organ lesions and necrotising pneumonia, i.e., clinical manifestations resembling symptoms of severe PVL-associated disease in humans. It might thus be assumed that the "Beaver Leukocidin (BVL, lukF/S-BV)"-positive strains are beaver-specific pathogens, and further studies on their clinical role as well as on a possible transmissibility to other species, including humans, are warranted.


Assuntos
Toxinas Bacterianas/análise , Exotoxinas/análise , Leucocidinas/análise , Roedores/microbiologia , Infecções Estafilocócicas/microbiologia , Fagos de Staphylococcus/isolamento & purificação , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/virologia , Animais , Toxinas Bacterianas/genética , Técnicas de Tipagem Bacteriana , Exotoxinas/genética , Genes Bacterianos , Genes Virais , Humanos , Leucocidinas/genética , Infecções Estafilocócicas/veterinária , Fagos de Staphylococcus/genética , Staphylococcus aureus/genética
20.
Viruses ; 13(11)2021 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-34834951

RESUMO

Understanding the evolution of viral pathogens is critical to being able to define how viruses emerge within different landscapes. Host susceptibility, which is spread between different species and is a contributing factor to the subsequent epidemiology of a disease, is defined by virus detection and subsequent characterization. Peste des petits ruminants virus is a plague of small ruminant species that is a considerable burden to the development of sustainable agriculture across Africa and much of Asia. The virus has also had a significant impact on populations of endangered species in recent years, highlighting its significance as a pathogen of high concern across different regions of the globe. Here, we have re-evaluated the molecular evolution of this virus using novel genetic data to try and further resolve the molecular epidemiology of this disease. Viral isolates are genetically characterized into four lineages (I-IV), and the historic origin of these lineages is of considerable interest to the molecular evolution of the virus. Our re-evaluation of viral emergence using novel genome sequences has demonstrated that lineages I, II and IV likely originated in West Africa, in Senegal (I) and Nigeria (II and IV). Lineage III sequences predicted emergence in either East Africa (Ethiopia) or in the Arabian Peninsula (Oman and/or the United Arab Emirates), with a paucity of data precluding a more refined interpretation. Continual refinements of evolutionary emergence, following the generation of new data, is key to both understanding viral evolution from a historic perspective and informing on the ongoing genetic emergence of this virus.


Assuntos
Evolução Molecular , Genes Virais , Peste dos Pequenos Ruminantes/epidemiologia , Peste dos Pequenos Ruminantes/virologia , Vírus da Peste dos Pequenos Ruminantes/classificação , Vírus da Peste dos Pequenos Ruminantes/genética , África Oriental/epidemiologia , África Ocidental/epidemiologia , Animais , Ásia/epidemiologia , Surtos de Doenças , Etiópia/epidemiologia , Genoma Viral , Doenças das Cabras/virologia , Cabras/virologia , Epidemiologia Molecular , Filogenia , Ruminantes/virologia , Senegal/epidemiologia , Análise de Sequência de DNA , Emirados Árabes Unidos/epidemiologia , Sequenciamento Completo do Genoma
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