RESUMO
Adeno-associated viruses (AAVs) package a single-stranded (ss) DNA genome of 4.7 kb in their capsid of ~20 nm in diameter. AAV replication requires co-infection of a helper virus, such as adenovirus. During the optimization of recombinant AAV production, a small viral nonstructural protein, membrane-associated accessory protein (MAAP), was identified. However, the function of the MAAP in the context of AAV infection remains unknown. Here, we investigated the expression strategy and function of the MAAP during infection of both AAV2 and AAV5 in human embryonic kidney (HEK)293 cells. We found that AAV2 MAAP2 and AAV5 MAAP5 are expressed from the capsid gene (cap)-transcribing mRNA spliced from the donor to the second splice site that encodes VP2 and VP3. Thus, this AAV cap gene transcribes a multicistronic mRNA that can be translated to four viral proteins, MAAP, VP2, AAP, and VP3 in order. In AAV2 infection, MAAP2 predominantly localized in the cytoplasm, alongside the capsid, near the nuclear and plasma membranes, but a fraction of MAAP2 exhibited nuclear localization. In AAV5 infection, MAAP5 revealed a distinct pattern, predominantly localizing within the nucleus. In the cells infected with an MAAP knockout mutant of AAV2 or AAV5, both viral DNA replication and virus replication increased, whereas virus egress decreased, and the decrease in virus egress can be restored by providing MAAP in trans. In summary, MAAP, a novel AAV nonstructural protein translated from a multicistronic viral cap mRNA, not only facilitates cellular egress of AAV but also likely negatively affects viral DNA replication during infection. IMPORTANCE: Recombinant adeno-associated virus (rAAV) has been used as a gene delivery vector in clinical gene therapy. In current gene therapies employing rAAV, a high dose of the vector is required. Consequently, there is a high demand for efficient and high-purity vector production systems. In this study, we demonstrated that membrane-associated accessory protein (MAAP), a small viral nonstructural protein, is translated from the same viral mRNA transcript encoding VP2 and VP3. In AAV-infected cells, apart from its prevalent expression in the cytoplasm with localization near the plasma and nuclear membranes, the MAAP also exhibits notable localization within the nucleus. During AAV infection, MAAP expression increases the cellular egress of progeny virions and decreases viral DNA replication and progeny virion production. Thus, the choice of MAAP expression has pros and cons during AAV infection, which could provide a guide to rAAV production.
Assuntos
Dependovirus , Infecções por Parvoviridae , Proteínas não Estruturais Virais , Humanos , Proteínas do Capsídeo/genética , Dependovirus/genética , Dependovirus/metabolismo , Dependovirus/fisiologia , Células HEK293 , Infecções por Parvoviridae/virologia , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Replicação Viral , Genes Virais/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.
Assuntos
Genoma Viral , Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Mutação , RNA Polimerase II , Transcrição Viral , Animais , Humanos , Linhagem Celular , Chlorocebus aethiops , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Genes Virais/genética , Genoma Viral/genética , Herpes Simples/virologia , Herpes Simples/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/genética , Poli A/genética , Poli A/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Células Vero , Transcrição Viral/efeitos dos fármacos , Transcrição Viral/genética , Replicação Viral/genéticaRESUMO
Viral genomes frequently harbor overlapping genes, complicating the development of virus-vectored vaccines and gene therapies. This study introduces a novel conditional splicing system to precisely control the expression of such overlapping genes through recombinase-mediated conditional splicing. We refined site-specific recombinase (SSR) conditional splicing systems and explored their mechanisms. The systems demonstrated exceptional inducibility (116,700-fold increase) with negligible background expression, facilitating the conditional expression of overlapping genes in adenovirus-associated virus (AAV) and human immunodeficiency virus type 1. Notably, this approach enabled the establishment of stable AAV producer cell lines, encapsulating all necessary packaging genes. Our findings underscore the potential of the SSR-conditional splicing system to significantly advance vector engineering, enhancing the efficacy and scalability of viral-vector-based therapies and vaccines. IMPORTANCE: Regulating overlapping genes is vital for gene therapy and vaccine development using viral vectors. The regulation of overlapping genes presents challenges, including cytotoxicity and impacts on vector capacity and genome stability, which restrict stable packaging cell line development and broad application. To address these challenges, we present a "loxp-splice-loxp"-based conditional splicing system, offering a novel solution for conditional expression of overlapping genes and stable cell line establishment. This system may also regulate other cytotoxic genes, representing a significant advancement in cell engineering and gene therapy as well as biomass production.
Assuntos
Dependovirus , Homologia de Genes , Genes Virais , Engenharia Genética , HIV-1 , Splicing de RNA , Humanos , Linhagem Celular , Dependovirus/genética , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Regulação Viral da Expressão Gênica , Homologia de Genes/genética , Genes Virais/genética , Engenharia Genética/métodos , Terapia Genética/métodos , Vetores Genéticos/genética , HIV-1/genética , Splicing de RNA/genética , Vacinas/biossíntese , Vacinas/genética , Empacotamento do Genoma Viral/genéticaRESUMO
BACKGROUND: Human cytomegalovirus (HCMV) is the most common and serious opportunistic infection after solid organ and hematopoietic stem cell transplantation. In this study, we used whole-genome HCMV data to investigate viral factors associated with the clinical outcome. METHODS: We sequenced HCMV samples from 16 immunocompromised pediatric patients with persistent viremia. Eight of the 16 patients died of complications due to HCMV infection. We also sequenced samples from 35 infected solid organ adult recipients, of whom 1 died with HCMV infection. RESULTS: We showed that samples from both groups have fixed variants at resistance sites and mixed infections. Next-generation sequencing also revealed nonfixed variants at resistance sites in most of the patients who died (6/9). A machine learning approach identified 10 genes with nonfixed variants in these patients. These genes formed a viral signature that discriminated patients with HCMV infection who died from those who survived with high accuracy (area under the curve = 0.96). Lymphocyte numbers for a subset of patients showed no recovery posttransplant in the patients who died. CONCLUSIONS: We hypothesize that the viral signature identified in this study may be a useful biomarker for poor response to antiviral drug treatment and indirectly for poor T-cell function, potentially identifying early those patients requiring nonpharmacological interventions.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Hospedeiro Imunocomprometido , Humanos , Infecções por Citomegalovirus/virologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/mortalidade , Citomegalovirus/genética , Criança , Masculino , Feminino , Adulto , Pré-Escolar , Antivirais/uso terapêutico , Adolescente , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Lactente , Transplante de Órgãos/efeitos adversos , Sequenciamento de Nucleotídeos em Larga Escala , Viremia , Genes Virais/genética , Variação Genética , Farmacorresistência Viral/genética , Adulto Jovem , Pessoa de Meia-IdadeRESUMO
TarBase is a reference database dedicated to produce, curate and deliver high quality experimentally-supported microRNA (miRNA) targets on protein-coding transcripts. In its latest version (v9.0, https://dianalab.e-ce.uth.gr/tarbasev9), it pushes the envelope by introducing virally-encoded miRNAs, interactions leading to target-directed miRNA degradation (TDMD) events and the largest collection of miRNA-gene interactions to date in a plethora of experimental settings, tissues and cell-types. It catalogues â¼6 million entries, comprising â¼2 million unique miRNA-gene pairs, supported by 37 experimental (high- and low-yield) protocols in 172 tissues and cell-types. Interactions are annotated with rich metadata including information on genes/transcripts, miRNAs, samples, experimental contexts and publications, while millions of miRNA-binding locations are also provided at cell-type resolution. A completely re-designed interface with state-of-the-art web technologies, incorporates more features, and allows flexible and ingenious use. The new interface provides the capability to design sophisticated queries with numerous filtering criteria including cell lines, experimental conditions, cell types, experimental methods, species and/or tissues of interest. Additionally, a plethora of fine-tuning capacities have been integrated to the platform, offering the refinement of the returned interactions based on miRNA confidence and expression levels, while boundless local retrieval of the offered interactions and metadata is enabled.
Assuntos
Bases de Dados de Ácidos Nucleicos , MicroRNAs , Genes Virais/genética , Internet , MicroRNAs/genética , MicroRNAs/metabolismo , AnimaisRESUMO
The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic that emerged in 2019 has been an unprecedented event in international science, as it has been possible to sequence millions of genomes, tracking their evolution very closely. This has enabled various types of secondary analyses of these genomes, including the measurement of their sequence selection pressure. In this work, we have been able to measure the selective pressure of all the described SARS-CoV-2 genes, even analyzed by sequence regions, and we show how this type of analysis allows us to separate the genes between those subject to positive selection (usually those that code for surface proteins or those exposed to the host immune system) and those subject to negative selection because they require greater conservation of their structure and function. We have also seen that when another gene with an overlapping reading frame appears within a gene sequence, the overlapping sequence between the two genes evolves under a stronger purifying selection than the average of the non-overlapping regions of the main gene. We propose this type of analysis as a useful tool for locating and analyzing all the genes of a viral genome when an adequate number of sequences are available.IMPORTANCEWe have analyzed the selection pressure of all severe acute respiratory syndrome coronavirus 2 genes by means of the nonsynonymous (Ka) to synonymous (Ks) substitution rate. We found that protein-coding genes are exposed to strong positive selection, especially in the regions of interaction with other molecules (host receptor and genome of the virus itself). However, overlapping coding regions are more protected and show negative selection. This suggests that this measure could be used to study viral gene function as well as overlapping genes.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Proteínas , Genoma Viral/genética , Genes Virais/genéticaRESUMO
IMPORTANCE: African swine fever (ASF) caused by ASF virus (ASFV) is a highly contagious and acute hemorrhagic viral disease in domestic pigs. Until now, no effective commercial vaccine and antiviral drugs are available for ASF control. Here, we generated a new live-attenuated vaccine candidate (ASFV-ΔH240R-Δ7R) by deleting H240R and MGF505-7R genes from the highly pathogenic ASFV HLJ/18 genome. Piglets immunized with ASFV-ΔH240R-Δ7R were safe without any ASF-related signs and produced specific antibodies against p30. Challenged with a virulent ASFV HLJ/18, the piglets immunized with high-dose group (105 HAD50) exhibited 100% protection without clinical symptoms, showing that low levels of virus replication with no observed pathogenicity by postmortem and histological analysis. Overall, our results provided a new strategy by designing live-attenuated vaccine candidate, resulting in protection against ASFV infection.
Assuntos
Vírus da Febre Suína Africana , Deleção de Genes , Genes Virais , Vacinas Atenuadas , Vacinas Virais , Animais , Febre Suína Africana/imunologia , Febre Suína Africana/prevenção & controle , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/classificação , Vírus da Febre Suína Africana/imunologia , Vírus da Febre Suína Africana/patogenicidade , Sus scrofa/virologia , Vacinas Atenuadas/imunologia , Proteínas Virais/genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência , Replicação Viral , Genes Virais/genéticaRESUMO
IMPORTANCE: Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Transcrição Gênica , Humanos , Genes Virais/genética , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/metabolismo , Iniciação da Transcrição Genética , Elongação da Transcrição Genética , Terminação da Transcrição GenéticaRESUMO
The African swine fever virus (ASFV) has caused a devastating pandemic in domestic and wild swine, causing economic losses to the global swine industry. Recombinant live attenuated vaccines are an attractive option for ASFV treatment. However, safe and effective vaccines against ASFV are still scarce, and more high-quality experimental vaccine strains need to be developed. In this study, we revealed that deletion of the ASFV genes DP148R, DP71L, and DP96R from the highly virulent isolate ASFV CN/GS/2018 (ASFV-GS) substantially attenuated virulence in swine. Pigs infected with 104 50% hemadsorbing doses of the virus with these gene deletions remained healthy during the 19-day observation period. No ASFV infection was detected in contact pigs under the experimental conditions. Importantly, the inoculated pigs were protected against homologous challenges. Additionally, RNA sequence analysis showed that deletion of these viral genes induced significant upregulation of the host histone H3.1 gene (H3.1) and downregulation of the ASFV MGF110-7L gene. Knocking down the expression of H3.1 resulted in high levels of ASFV replication in primary porcine macrophages in vitro. These findings indicate that the deletion mutant virus ASFV-GS-Δ18R/NL/UK is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce full protection against the highly virulent ASFV-GS virus strain. IMPORTANCE Ongoing outbreaks of African swine fever (ASF) have considerably damaged the pig industry in affected countries. Thus, a safe and effective vaccine is important to control African swine fever spread. Here, an ASFV strain with three gene deletions was developed by knocking out the viral genes DP148R (MGF360-18R), NL (DP71L), and UK (DP96R). The results showed that the recombinant virus was completely attenuated in pigs and provided strong protection against parental virus challenge. Additionally, no viral genomes were detected in the sera of pigs housed with animals infected with the deletion mutant. Furthermore, transcriptome sequencing (RNA-seq) analysis revealed significant upregulation of histone H3.1 in virus-infected macrophage cultures and downregulation of the ASFV MGF110-7L gene after viral DP148R, UK, and NL deletion. Our study provides a valuable live attenuated vaccine candidate and potential gene targets for developing strategies for anti-ASFV treatment.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Deleção de Genes , Genes Virais , Vacinas Virais , Fatores de Virulência , Animais , Febre Suína Africana/imunologia , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/imunologia , Vírus da Febre Suína Africana/patogenicidade , Células Cultivadas , Genes Virais/genética , Histonas/genética , Suínos , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Fatores de Virulência/genéticaRESUMO
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 , HumanosRESUMO
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/genéticaRESUMO
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 , Povo Asiático , 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/genéticaRESUMO
Compatibility among the influenza A virus (IAV) ribonucleoprotein (RNP) genes affects viral replication efficiency and can limit the emergence of novel reassortants, including those with potential pandemic risks. In this study, we determined the polymerase activities of 2,451 RNP reassortants among three seasonal and eight enzootic IAVs by using a minigenome assay. Results showed that the 2009 H1N1 RNP are more compatible with the tested enzootic RNP than seasonal H3N2 RNP and that triple reassortment increased such compatibility. The RNP reassortants among 2009 H1N1, canine H3N8, and avian H4N6 IAVs had the highest polymerase activities. Residues in the RNA binding motifs and the contact regions among RNP proteins affected polymerase activities. Our data indicates that compatibility among seasonal and enzootic RNPs are selective, and enzoosis of multiple strains in the animal-human interface can facilitate emergence of an RNP with increased replication efficiency in mammals, including humans.
Assuntos
Genes Virais/genética , Vírus da Influenza A/genética , Infecções por Orthomyxoviridae/genética , Vírus Reordenados/genética , Ribonucleoproteínas/genética , Animais , HumanosRESUMO
Varicella-zoster virus (VZV) is a human herpes virus which causes varicella (chicken pox) as a primary infection, and, following a variable period of latency in neurons in the peripheral ganglia, may reactivate to cause herpes zoster (shingles) as well as a variety of neurological syndromes. In this overview we consider some recent issues in alphaherpesvirus latency with special focus on VZV ganglionic latency. A key question is the nature and extent of viral gene transcription during viral latency. While it is known that this is highly restricted, it is only recently that the very high degree of that restriction has been clarified, with both VZV gene 63-encoded transcripts and discovery of a novel VZV transcript (VLT) that maps antisense to the viral transactivator gene 61. It has also emerged in recent years that there is significant epigenetic regulation of VZV gene transcription, and the mechanisms underlying this are complex and being unraveled. The last few years has also seen an increased interest in the immunological aspects of VZV latency and reactivation, in particular from the perspective of inborn errors of host immunity that predispose to different VZV reactivation syndromes.
Assuntos
Herpesvirus Humano 3/metabolismo , Infecção pelo Vírus da Varicela-Zoster/genética , Latência Viral/genética , Varicela/virologia , Epigênese Genética/genética , Genes Virais/genética , Herpes Zoster/virologia , Herpesvirus Humano 3/patogenicidade , Humanos , Neurônios/virologia , Infecção pelo Vírus da Varicela-Zoster/epidemiologia , Latência Viral/fisiologiaRESUMO
African swine fever (ASF) is a severe hemorrhagic infectious disease in pigs caused by African swine fever virus (ASFV), leading to devastating economic losses in epidemic regions. Its control currently depends on thorough culling and clearance of the diseased and surrounding suspected pigs. An ASF vaccine has been extensively explored for years worldwide, especially in hog-intensive areas where it is highly desired, but it is still unavailable for numerous reasons. Here, we report another ASF vaccine candidate, named SY18ΔI226R, bearing a deletion of the I226R gene with a replacement of an enhanced green fluorescent protein (eGFP) expression cassette at the right end of the viral genome. This deletion results in the complete loss of virulence of SY18 as the gene-deleted strain does not cause any clinical symptoms in all pigs inoculated with a dosage of either 104.0 or 107.0 50% tissue culture infective doses (TCID50). Apparent viremia with a gradual decline was monitored, while virus shedding was detected only occasionally in oral or anal swabs. ASFV-specific antibody appeared at 9 days postinoculation. After intramuscular challenge with its parental strain ASFV SY18 at 21 days postinoculation, all the challenged pigs survived, without obvious febrile or abnormal clinical signs. No viral DNA could be detected upon the dissection of any tissue when viremia disappeared. These results indicated that SY18ΔI226R is safe in swine and elicits robust immunity to virulent ASFV infection. IMPORTANCE Outbreaks of African swine fever have resulted in devastating losses to the swine industry worldwide, but there is currently no commercial vaccine available. Although several vaccine candidates have been reported, none has been approved for use for several reasons, especially ones concerning biosafety. Here, we identified a new undescribed functional gene, I226R. When deleted from the ASFV genome, the virus completely loses its virulence in swine. Importantly, pigs infected with this gene-deleted virus were resistant to infection by intramuscular challenge with 102.5 or 104.0 TCID50 of its virulent parental virus. Furthermore, the nucleic acid of the gene-deleted virus and its virulent parental virus was rarely detected from oral or anal swabs. Viruses could not be detected in any tissues after necropsy when viremia became negative, indicating that robust immunity was achieved. Therefore, SY18ΔI226R is a novel, ideal, and efficacious vaccine candidate for genotype II ASF.
Assuntos
Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/imunologia , Febre Suína Africana/imunologia , Deleção de Genes , Genoma Viral , Febre Suína Africana/patologia , Febre Suína Africana/prevenção & controle , Animais , DNA Viral , Genes Virais/genética , Genótipo , Análise de Sequência , Suínos , Vacinas Virais/imunologia , Viremia/genética , Virulência/genéticaRESUMO
BACKGROUND: Coronavirus disease of 2019 (COVID-19) is well known as a fatal disease, first discovered at Wuhan in China, ranging from mild to death, such as shortness of breath and fever. Early diagnosis of COVID-19 is a crucial point in preventing global prevalence. OBJECTIVE: We aimed to evaluate the diagnostic competency and efficiency with the Allplex™ 2019-nCoV Assay kit and the Dr. PCR 20 K COVID-19 Detection kit, designed based on the qRT-PCR and dPCR technologies, respectively. METHODS: A total of 30 negative and 20 COVID-19 positive specimens were assigned to the diagnostic test by using different COVID-19 diagnosis kits. Diagnostic accuracy was measured by statistical testing with sensitivity, specificity, and co-efficiency calculations. RESULTS: Comparing both diagnostic kits, we confirmed that the diagnostic results of 30 negative and 20 positive cases were the same pre-diagnostic results. The diagnostic statistics test results were perfectly matched with value (1). Cohen's Kappa coefficient was demonstrated that the given kits in two different ways were "almost perfect" with value (1). In evaluating the detection capability, the dilutional linearity experiments substantiate that the Dr. PCR 20 K COVID-19 Detection kit could detect SARS-CoV-2 viral load at a concentration ten times lower than that of the Allplex™ 2019-nCoV Assay kit. CONCLUSIONS: In this study, we propose that the dPCR diagnosis using LOAA dPCR could be a powerful method for COVID-19 point-of-care tests requiring immediate diagnosis in a limited time and space through the advantages of relatively low sample concentration and small equipment size compared to conventional qRT-PCR.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/isolamento & purificação , COVID-19 , Genes Virais/genética , Humanos , República da Coreia , SARS-CoV-2/genética , Sensibilidade e Especificidade , Carga ViralRESUMO
As one of the largest biotechnological applications, activated sludge (AS) systems in wastewater treatment plants (WWTPs) harbor enormous viruses, with 10-1,000-fold higher concentrations than in natural environments. However, the compositional variation and host-connections of AS viruses remain poorly explored. Here, we report a catalogue of ~50,000 prokaryotic viruses from six WWTPs, increasing the number of described viral species of AS by 23-fold, and showing the very high viral diversity which is largely unknown (98.4-99.6% of total viral contigs). Most viral genera are represented in more than one AS system with 53 identified across all. Viral infection widely spans 8 archaeal and 58 bacterial phyla, linking viruses with aerobic/anaerobic heterotrophs, and other functional microorganisms controlling nitrogen/phosphorous removal. Notably, Mycobacterium, notorious for causing AS foaming, is associated with 402 viral genera. Our findings expand the current AS virus catalogue and provide reference for the phage treatment to control undesired microorganisms in WWTPs.
Assuntos
Ciclo do Carbono , Células Procarióticas/virologia , Esgotos/virologia , Viroma/genética , Vírus/genética , Purificação da Água/métodos , Archaea/classificação , Archaea/genética , Archaea/virologia , Bactérias/classificação , Bactérias/genética , Bactérias/virologia , Metabolismo Energético/genética , Genes Virais/genética , Variação Genética , Interações Hospedeiro-Patógeno , Fases de Leitura Aberta/genética , Células Procarióticas/metabolismo , Análise de Sequência de DNA/métodos , Esgotos/microbiologia , Vírus/classificação , Vírus/metabolismoRESUMO
Senecavirus A (SVA), formerly known as Seneca Valley virus, is classified into the genus Senecavirus in the family Picornaviridae. Mature virion harbors an approximately 7 300-nt-long, positive-sense, and single-stranded RNA genome, which contains 5' and 3' untranslated regions (UTRs). Internal ribosome entry site (IRES) is identified in the SVA 5' UTR, and includes a RNA pseudoknot upstream of the start codon. This pseudoknot contains two stem structures, pseudoknot stem I and II (PKS-I and -II). The PKS-I is composed of two base-paired motifs (PKS-Ia and -Ib), between which there is an unpaired spacing (UpS). We reported previously that motif mutation in the PKS-II did not abolish the IRES activity, but interfered with SVA recovery from cDNA clone. In this study, we constructed five SVA minigenomes with point mutations in the PKS-I motif. Dual-luciferase reporter assay showed that motif mutations in PKS-I did not significantly interfere with the IRES activity to initiate protein expression. Correspondingly, we constructed five SVA cDNA clones with point mutations in the PKS-I motif. These genetically modified cDNA clones were separately transfected into BSR-T7/5 cells in attempting to rescue competent SVAs. However, only two viruses, namely PKS-Ia- and UpS-mutated recombinants, could be recovered from their individual cDNA clones. It can be concluded that the PKS-Ib is indispensable for viral growth.
Assuntos
Códon de Iniciação , Sítios Internos de Entrada Ribossomal , Picornaviridae , Animais , Códon de Iniciação/genética , DNA Complementar , Genes Virais/genética , Mutação , Conformação de Ácido Nucleico , Picornaviridae/genética , RNA Viral/genéticaRESUMO
African swine fever virus (ASFV) is a large nucleoplasmic DNA virus, in which the genome is around 170-198 kilobases (kb). More than 50 % genes have unknown functions. Here, MGF100-1R gene is chosen to study the primary function and sublocalization. The gene was located at the left variable region of the ASFV genome that belongs to MGF100 families. It located at the cytoplasm without cytotoxic activities. However, it related to induce the transcriptional levels of pro-inflammatory cytokines. A deletion mutant of MGF100-1R gene was constructed based on ASFV Chinese strain GZ201801. The recombinant deletion mutant (ASFVâ³MGF100-1R) was demonstrated in vitro that the gene is non-essential for virus replication with a similar replication kinetics in bone marrow-derived macrophages (BMDMs) cell cultures when compared to parental virus. In vivo evaluation, ASFVâ³MGF100-1R was inoculated intramuscularly and led to a similar pathogenesis that caused by the parental ASFV GZ201801, confirming that deletion of MGF100-1R gene from the ASFV genome does not impact virulence.
Assuntos
Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/patogenicidade , Febre Suína Africana/virologia , Deleção de Sequência/genética , Animais , Células Cultivadas , China , Genes Virais/genética , Macrófagos/virologia , Suínos , Virulência/genética , Replicação Viral/genéticaRESUMO
Porcine circovirus type 3 (PCV3) is a highly contagious virus belonging to the family Circoviridae that causes the severe dermatitis and nephropathy syndrome. To date, PCV3 has a worldwide distribution and bring huge economic losses to swine industry. Replicase (Rep) and capsid (Cap) are two major coded proteins of PCV3. Considering the large number of new PCV3 isolates were reported in the past few years and the research for the codon usage pattern of Rep and Cap genes was still a gap, phylogenetic and codon usage analysis of these two genes was performed. Phylogenetic analyses showed that Rep genes in PCV3a were dispersed with no clear clusters while corresponding sequences in PCV3b clustered into two groups and Cap genes clustered into distinct clades according to different genotypes. Relative synonymous codon usage (RSCU) analysis revealed that the codon usage bias existed and effective number of codon (ENC) analysis showed that the bias was slight low. ENC-GC3s plot indicated that mutational pressure and other factors both played a role in PCV3 codon usage and neutrality plot analysis showed that natural selection was the main force influencing the codon usage pattern. The results presented here provided the important basic data on codon usage pattern of Rep and Cap genes, and a better understanding of the evolution and potential origin of PCV3.