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1.
Sci Rep ; 11(1): 5372, 2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33686189

RESUMO

Wastewater-based epidemiology (WBE) is a great approach that enables us to comprehensively monitor the community to determine the scale and dynamics of infections in a city, particularly in metropolitan cities with a high population density. Therefore, we monitored the time course of the SARS-CoV-2 RNA concentration in raw sewage in the Frankfurt metropolitan area, the European financial center. To determine the SARS-CoV-2 RNA concentration in sewage, we continuously collected 24 h composite samples twice a week from two wastewater treatment plant (WWTP) influents (Niederrad and Sindlingen) serving the Frankfurt metropolitan area and performed RT-qPCR analysis targeting three genes (N gene, S gene, and ORF1ab gene). In August, a resurgence in the SARS-CoV-2 RNA load was observed, reaching 3 × 1013 copies/day, which represented similar levels compared to April with approx. 2 × 1014 copies/day. This corresponds to a continuous increase again in COVID-19 cases in Frankfurt since August, with an average of 28.6 incidences, compared to 28.7 incidences in April. Different temporal dynamics were observed between different sampling points, indicating local dynamics in COVID-19 cases within the Frankfurt metropolitan area. The SARS-CoV-2 RNA load to the WWTP Niederrad ranged from approx. 4 × 1011 to 1 × 1015 copies/day, the load to the WWTP Sindlingen from approx. 1 × 1011 to 2 × 1014 copies/day, which resulted in a preceding increase in these loading in July ahead of the weekly averaged incidences. The study shows that WBE has the potential as an early warning system for SARS-CoV-2 infections and a monitoring system to identify global hotspots of COVID-19.


Assuntos
Monitoramento Ambiental , RNA Viral/análise , Águas Residuárias/virologia , /epidemiologia , Cidades , Monitoramento Epidemiológico , Genes Virais , Alemanha , Esgotos/virologia , Fatores de Tempo , Carga Viral , Purificação da Água
2.
Int J Hyperthermia ; 38(1): 202-212, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33682604

RESUMO

Increased transmissibility of the pandemic severe acute respiratory coronavirus 2 (SARS-CoV-2) has been noted to occur at lower ambient temperatures. This is seemingly related to a better replication of most respiratory viruses, including SARS-CoV-2, at lower-than-core body temperatures (i.e., 33 °C vs 37 °C). Also, intrinsic characteristics of SARS-CoV-2 make it a heat-susceptible pathogen. Thermotherapy has successfully been used to combat viral infections in plants which could otherwise result in great economic losses; 90% of viruses causing infections in plants are positive-sense single-stranded ribonucleic acid (+ssRNA) viruses, a characteristic shared by SARS-CoV-2. Thus, it is possible to envision the use of heat-based interventions (thermotherapy or mild-temperature hyperthermia) in patients with COVID-19 for which moderate cycles (every 8-12 h) of mild-temperature hyperthermia (1-2 h) have been proposed. However, there are potential safety and mechanistic concerns which could limit the use of thermotherapy only to patients with mild-to-moderate COVID-19 to prevent disease progression rather than to treat patients who have already progressed to severe-to-critical COVID-19. Here, we review the characteristics of SARS-CoV-2 which make it a heat-susceptible virus, potential host mechanisms which could be enhanced at higher temperatures to aid viral clearance, and how thermotherapy could be investigated as a modality of treatment in patients with COVID-19 while taking into consideration potential risks.


Assuntos
/terapia , Hipertermia Induzida , Animais , Temperatura Corporal , Genes Virais , Humanos , Plantas/virologia , Interferência de RNA , /isolamento & purificação
3.
Biochem Biophys Res Commun ; 550: 8-14, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33676232

RESUMO

The SARS-CoV-2 Variant of Concern 202012/01 (VOC-202012/01) emerged in southeast England and rapidly spread worldwide. This variant is believed to be more transmissible, with all attention being given to its spike mutations. However, VOC-202012/01 has also a mutation (Q27stop) that truncates the ORF8, a likely immune evasion protein. Removal of ORF8 changes the clinical outset of the disease, which may affect the virus transmissibility. Here I provide a detailed analysis of all reported ORF8-deficient lineages found in the background of relevant spike mutations, identified among 231,433 SARS-CoV-2 genomes. I found 19 ORF8 nonsense mutations, most of them occurring in the 5' half of the gene. The ORF8-deficient lineages were rare, representing 0.67% of sequenced genomes. Nevertheless, I identified two clusters of related sequences that emerged recently and spread in different countries. The widespread D614G spike mutation was found in most ORF-deficient lineages. Although less frequent, HV69-70del and L5F spike mutations occurred in the background of six different ORF8 nonsense mutations. I also confirmed that VOC-202012/01 is the ORF8-deficient variant with more spike mutations reported to date, although other variants could have up to six spike mutations, some of putative biological relevance. Overall, these results suggest that monitoring ORF8-deficient lineages is important for the progression of the COVID-19 pandemic, particularly when associated with relevant spike mutations.


Assuntos
/transmissão , Monitoramento Epidemiológico , Deleção de Genes , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais/genética , /epidemiologia , Códon sem Sentido , Códon de Terminação/genética , Evolução Molecular , Genes Virais/genética , Humanos , Filogenia , Seleção Genética , Fatores de Tempo , Reino Unido/epidemiologia
4.
Dokl Biochem Biophys ; 496(1): 27-31, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33689070

RESUMO

The coronavirus family consists of lipid-containing envelope viruses that have a single-stranded RNA genome that encodes 25-30 proteins in different viruses by the mechanism of positive-polarity strategy. In addition, extended open reading trnslation frames (ORFs, genes) located in a negative-sense orientation were found in the genomes of coronaviruses. The size of negative-sense genes varies in the range of 150-450 nt, which corresponds to polypeptides encoded by negative-polarity genes (negative gene proteins, NGP) with m. m. 5-30 × 103 kDa. Coronaviruses show marked differences from virus to virus in the number of negative genes detected. These negative-sense genes in the coronavirus genome allow this family to be considered as viruses developing an ambisense genome strategy.


Assuntos
Genes Virais/genética , Genômica , RNA Viral/genética , /genética , Sequência de Bases , Fases de Leitura Aberta/genética
5.
Methods Mol Biol ; 2244: 133-158, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555586

RESUMO

To fully understand the function of cytomegalovirus (CMV) genes, it is imperative that they are studied in the context of infection. Therefore, the targeted deletion of individual viral genes and the comparison of these loss-of-function viral mutants to the wild-type virus allow for the identification of the relevance and role for a particular gene in the viral replication cycle. Targeted CMV mutagenesis has made huge advances over the past 20 years. The cloning of CMV genomes into Escherichia coli as bacterial artificial chromosomes (BAC) allows for not only quick and efficient deletion of viral genomic regions, individual genes, or single-nucleotide exchanges in the viral genome but also the insertion of heterologous genetic sequences for gain-of-function approaches. The conceptual advantage of this strategy is that it overcomes the restrictions of recombinant technologies in cell culture systems. Namely, recombination in infected cells occurs only in a few clones, and their selection is not possible if the targeted genes are relevant for virus replication and are not able to compete for growth against the unrecombined parental viruses. On the other hand, BAC mutagenesis enables the selection for antibiotic resistance in E. coli, providing selective growth advantage to the recombined genomes and thus clonal selection of viruses with even extremely poor fitness. Here we describe the methods used for the generation of a CMV BAC, targeted mutagenesis of BAC clones, and transfection of human cells with CMV BAC DNA in order to reconstitute the viral infection process.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Clonagem Molecular/métodos , Citomegalovirus/genética , Células Cultivadas , Escherichia coli/genética , Genes Virais/genética , Genoma Viral/genética , Humanos , Mutagênese/genética , Transfecção/métodos , Replicação Viral/genética
6.
Viruses ; 13(2)2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567491

RESUMO

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a major epidemic disease endangering the swine industry. Although a number of vaccine candidates have been reported, none are commercially available yet. To explore the effect of unknown genes on the biological characteristics of ASFV and the possibility of a gene-deleted isolate as a vaccine candidate, the strain SY18ΔL7-11, with deletions of L7L-L11L genes from ASFV SY18, was constructed, and its biological properties were analyzed. The results show that deletion of genes L7L-L11L did not affect replication of the virus in vitro. Virulence of SY18△L7-11 was significantly reduced, as 11 of the 12 pigs survived for 28 days after intramuscular inoculation with a low dose (103 TCID50) or a high dose (106 TCID50) of SY18ΔL7-11. All 11 surviving pigs were completely protected against challenge with the parental ASFV SY18 on 28 days postinoculation (dpi). Transient fever and/or irregularly low levels of genomic DNA in the blood were monitored in some pigs after inoculation. No ASF clinical signs or viremia were monitored after challenge. Antibodies to ASFV were induced in all pigs from 14 to 21 days postinoculation. IFN-γ was detected in most of the inoculated pigs, which is usually inhibited in ASFV-infected pigs. Overall, the results demonstrate that SY18ΔL7-11 is a candidate for further constructing safer vaccine(s), with better joint deletions of other gene(s) related to virulence.


Assuntos
Vírus da Febre Suína Africana/imunologia , Febre Suína Africana/prevenção & controle , Genes Virais/genética , Vacinas Virais/genética , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/patogenicidade , Animais , Anticorpos Antivirais/sangue , Células Cultivadas , Deleção de Genes , Injeções Intramusculares , Interferon gama/sangue , Macrófagos/virologia , Suínos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Virais/administração & dosagem , Virulência/genética
7.
Viruses ; 13(2)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33572446

RESUMO

Analysis of pooled genomic short read sequence data revealed the presence of nudivirus-derived sequences from U.S. populations of both southern corn rootworm (SCR, Diabrotica undecimpunctata howardi Barber) and western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte). A near complete nudivirus genome sequence was assembled from sequence data for an SCR population with relatively high viral titers. A total of 147,179 bp was assembled from five contigs that collectively encode 109 putative open reading frames (ORFs) including 20 nudivirus core genes. In contrast, genome sequence recovery was incomplete for a second nudivirus from WCR, although sequences derived from this virus were present in three geographically dispersed populations. Only 48,989 bp were assembled with 48 putative ORFs including 13 core genes, representing about 20% of a typical nudivirus genome. Phylogenetic analysis indicated that both corn rootworm nudiviruses grouped with the third known nudivirus of beetles, Oryctes rhinoceros nudivirus in the genus Alphanudivirus. On the basis of phylogenetic and additional analyses, we propose further taxonomic separation of nudiviruses within Alphanudivirus and Betanudivirus into two subfamilies and five genera. Identification of nudivirus-derived sequences from two species of corn rootworm highlights the diversity of viruses associated with these agricultural insect pests.


Assuntos
Besouros/virologia , Nudiviridae/genética , Animais , Besouros/classificação , DNA Viral/genética , Genes Virais , Genoma Viral/genética , Genômica , Nudiviridae/classificação , Fases de Leitura Aberta , Filogenia , /genética
8.
Sci Rep ; 11(1): 4108, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602998

RESUMO

In December 2019, rising pneumonia cases caused by a novel ß-coronavirus (SARS-CoV-2) occurred in Wuhan, China, which has rapidly spread worldwide, causing thousands of deaths. The WHO declared the SARS-CoV-2 outbreak as a public health emergency of international concern, since then several scientists are dedicated to its study. It has been observed that many human viruses have codon usage biases that match highly expressed proteins in the tissues they infect and depend on the host cell machinery for the replication and co-evolution. In this work, we analysed 91 molecular features and codon usage patterns for 339 viral genes and 463 human genes that consisted of 677,873 codon positions. Hereby, we selected the highly expressed genes from human lung tissue to perform computational studies that permit to compare their molecular features with those of SARS, SARS-CoV-2 and MERS genes. The integrated analysis of all the features revealed that certain viral genes and overexpressed human genes have similar codon usage patterns. The main pattern was the A/T bias that together with other features could propitiate the viral infection, enhanced by a host dependant specialization of the translation machinery of only some of the overexpressed genes. The envelope protein E, the membrane glycoprotein M and ORF7 could be further benefited. This could be the key for a facilitated translation and viral replication conducting to different comorbidities depending on the genetic variability of population due to the host translation machinery. This is the first codon usage approach that reveals which human genes could be potentially deregulated due to the codon usage similarities between the host and the viral genes when the virus is already inside the human cells of the lung tissues. Our work leaded to the identification of additional highly expressed human genes which are not the usual suspects but might play a role in the viral infection and settle the basis for further research in the field of human genetics associated with new viral infections. To identify the genes that could be deregulated under a viral infection is important to predict the collateral effects and determine which individuals would be more susceptible based on their genetic features and comorbidities associated.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Códon/genética , Uso do Códon , Biologia Computacional/métodos , Coronavirus/genética , Infecções por Coronavirus/metabolismo , Genes Virais , Genoma Viral , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Filogenia , Vírus da SARS/genética , /genética
9.
J Vet Sci ; 22(1): e12, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33522164

RESUMO

BACKGROUND: Bats have been considered natural reservoirs for several pathogenic human coronaviruses (CoVs) in the last two decades. Recently, a bat CoV was detected in the Republic of Korea; its entire genome was sequenced and reported to be genetically similar to that of the severe acute respiratory syndrome CoV (SARS-CoV). OBJECTIVES: The objective of this study was to compare the genetic sequences of SARS-CoV, SARS-CoV-2, and the two Korean bat CoV strains 16BO133 and B15-21, to estimate the likelihood of an interaction between the Korean bat CoVs and the human angiotensin-converting enzyme 2 (ACE2) receptor. METHODS: The phylogenetic analysis was conducted with the maximum-likelihood (ML) method using MEGA 7 software. The Korean bat CoVs receptor binding domain (RBD) of the spike protein was analyzed by comparative homology modeling using the SWISS-MODEL server. The binding energies of the complexes were calculated using PRODIGY and MM/GBGA. RESULTS: Phylogenetic analyses of the entire RNA-dependent RNA polymerase, spike regions, and the complete genome revealed that the Korean CoVs, along with SARS-CoV and SARS-CoV-2, belong to the subgenus Sarbecovirus, within BetaCoVs. However, the two Korean CoVs were distinct from SARS-CoV-2. Specifically, the spike gene of the Korean CoVs, which is involved in host infection, differed from that of SARS-CoV-2, showing only 66.8%-67.0% nucleotide homology and presented deletions within the RBD, particularly within regions critical for cross-species transmission and that mediate interaction with ACE2. Binding free energy calculation revealed that the binding affinity of Korean bat CoV RBD to hACE2 was drastically lower than that of SARS-CoV and SARS-CoV-2. CONCLUSIONS: These results suggest that Korean bat CoVs are unlikely to bind to the human ACE2 receptor.


Assuntos
Quirópteros/virologia , Coronavirus/genética , Vírus da SARS/genética , /genética , Animais , Genes Virais/genética , Genoma Viral/genética , Genômica , Humanos , Funções Verossimilhança , Filogenia , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , República da Coreia , Análise de Sequência de DNA , Homologia de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Ligação Viral
10.
Clin Lab ; 67(2)2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33616332

RESUMO

BACKGROUND AND METHODS: 2019 Corona Virus Disease (COVID-19) caused by SARS-CoV-2 is still pandemic now. RT-qPCR detection was the most common method for the diagnosis of SARS-CoV-2 infection, facilitated by amounts of nucleic acid testing kits. However, the accuracy of nucleic acid detection is affected by various factors such as specimen collection, specimen preparation, reagents deficiency, and personnel quality. RESULTS: In this study, we found that unmatched virus preservation solution will inhibit N gene and OFR-1ab gene (two independent genes of SARS-CoV-2) amplification in one-step detection reagent. CONCLUSIONS: Despite just being a particular phenomenon we found in our work to fight 2019-nCoV, we concluded that unmatched virus preservation solution may have an inhibitory effect on SARS-CoV-2 nucleic acid detection which may lead to incorrect clinical diagnosis.


Assuntos
/métodos , Genes Virais/efeitos dos fármacos , Soluções para Preservação de Órgãos/farmacologia , Manejo de Espécimes , /diagnóstico , Erros de Diagnóstico/prevenção & controle , Humanos , Kit de Reagentes para Diagnóstico/normas , /isolamento & purificação , Manejo de Espécimes/efeitos adversos , Manejo de Espécimes/métodos
11.
ACS Synth Biol ; 10(2): 379-390, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33534552

RESUMO

Generating and characterizing immunoreagents to enable studies of novel emerging viruses is an area where ensembles of synthetic genes, recombinant antibody pipelines, and modular antibody-reporter fusion proteins can respond rapidly. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread through the global population causing widespread morbidity, mortality, and socioeconomic chaos. Using SARS-CoV-2 as our model and starting with a gBlocks encoded nucleocapsid (N) gene, we purified recombinant protein from E. coli, to serve as bait for selecting semisynthetic nanobodies from our Nomad single-pot library. Clones were isolated in days and first fused to Gaussia luciferase to determine EC50 in the tens of nM range, and second fused to the ascorbate peroxidase derivative APEX2 for sensitive detection of SARS-CoV-2 infected cells. To generate inherently fluorescent immunoreagents, we introduce novel periplasmic sdAb fusions made with mNeonGreen and mScarlet-I, which were produced at milligram amounts. The fluorescent fusion proteins enabled concise visualization of SARS-CoV-2 N in the cytoplasm but not in the nucleus 24 h post infection, akin to the distribution of SARS-CoV N, thereby validating these useful imaging tools. SdAb reactivity appeared specific to SARS-CoV-2 with very much weaker binding to SARS-CoV, and no noticeable cross-reactivity to a panel of overexpressed human codon optimized N proteins from other CoV. High periplasmic expression levels and in silico immortalization of the nanobody constructs guarantees a cost-effective and reliable source of SARS-CoV-2 immunoreagents. Our proof-of-principle study should be applicable to known and newly emerging CoV to broaden the tools available for their analysis and help safeguard human health in a more proactive than reactive manner.


Assuntos
/epidemiologia , /genética , Sondas Moleculares/genética , Pandemias , /imunologia , Anticorpos Antivirais/genética , Especificidade de Anticorpos/genética , Doenças Transmissíveis Emergentes/virologia , Escherichia coli/genética , Imunofluorescência , Genes Sintéticos , Genes Virais , Células HEK293 , Humanos , Sondas Moleculares/imunologia , Pandemias/prevenção & controle , Biblioteca de Peptídeos , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Domínio Único/genética , Biologia Sintética
12.
Euro Surveill ; 26(5)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33541485

RESUMO

In June-November 2020, SARS-CoV-2-infected mink were detected in 290 of 1,147 Danish mink farms. In North Denmark Region, 30% (324/1,092) of people found connected to mink farms tested SARS-CoV-2-PCR-positive and approximately 27% (95% confidence interval (CI): 25-30) of SARS-CoV-2-strains from humans in the community were mink-associated. Measures proved insufficient to mitigate spread. On 4 November, the government ordered culling of all Danish mink. Farmed mink constitute a potential virus reservoir challenging pandemic control.


Assuntos
Animais Selvagens/virologia , /veterinária , Surtos de Doenças/veterinária , Reservatórios de Doenças/veterinária , Transmissão de Doença Infecciosa/veterinária , Vison/virologia , Pandemias/veterinária , /isolamento & purificação , /transmissão , Animais , /virologia , Dinamarca/epidemiologia , Surtos de Doenças/estatística & dados numéricos , Reservatórios de Doenças/virologia , Fazendas , Genes Virais , Humanos , Incidência , Reação em Cadeia da Polimerase , Saúde Pública , RNA Viral/análise , RNA Viral/genética , /virologia , Sequenciamento Completo do Genoma , Zoonoses/transmissão , Zoonoses/virologia
13.
Artigo em Inglês | MEDLINE | ID: mdl-33503149

RESUMO

Sexually transmitted infections (STIs) represent a global health problem with variable prevalence depending on the geographical region and the type of population. Human papillomavirus (HPV) encompasses widespread virus types related to cervical carcinogenesis. The present study investigated the molecular prevalence of HPV and seven other important STIs in asymptomatic women working or studying at a Brazilian university. A secondary aim was to assess cytological abnormalities associated with HPV and other STIs coinfections. We recruited 210 women from a Brazilian university. HPV was detected using a single-round polymerase chain reaction (sPCR) followed by a viral genotyping by restriction fragment length polymorphism (RFLP-PCR). The presence of seven STIs: Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, herpes simplex virus (HSV)-1 and HSV-2 was detected by multiplex PCR (M-PCR). Furthermore, cytological findings and epidemiological characteristics were evaluated.The mean age of the participants was 27.1 years old. HPV prevalence was 33.8%, and HPV16 was the most frequently detected papillomavirus genotype. Moreover, multiple HPV infections were common (42.2%). We detected at least one STI agent in 11.4% of the tested women, most frequently C. trachomatis (6.7%). Among HPV-positive women, 14.1% were coinfected with other STI agents. Cytological abnormalities were observed in 9.5% of smears, and HPV-DNA, high-risk HPV (HR-HPV), HPV16 and HPV multiple infections were associated with abnormal cytological findings. There was a high prevalence of HPV, and C. trachomatis was the most prevalent STI agent, with low rates of cytological abnormalities. These findings highlight the need of timely STI diagnosis in young asymptomatic women and of a public policy design for STI prevention.


Assuntos
Portador Sadio/epidemiologia , Papillomaviridae , Infecções por Papillomavirus/epidemiologia , Doenças Sexualmente Transmissíveis/epidemiologia , Adulto , Alphapapillomavirus , Brasil/epidemiologia , Feminino , Genes Virais , Genótipo , Humanos , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Prevalência , Universidades
14.
J Med Virol ; 93(4): 2538-2542, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33415765

RESUMO

The occurrence of the COVID-19 second-wave outbreak in Europe has pushed laboratories performing molecular SARS-CoV-2 tests to increase their throughput and decrease the result rendering time. In this evaluation, we tested for the first time a new, extraction-free, protocol with the Allplex SARS-CoV-2 Assay RT-qPCR kit on a Nimbus platform. Ninety-one samples, of which 71 previously tested positive with RT-qPCR with extraction were immediately analyzed without extraction, using only a dilution and thermal shock protocol. The positive and negative percentage agreements were respectively 97.2% (95% confidence interval [CI]: 0.90-0.99) and 95.0% (95% CI: 0.76-0.99). The two false negatives observed were very weakly positive with the comparison method. Moderate variations in Ct of the targeted genes were observed (median ± 95% CI): E gene, +2.49 ± 0.44; N gene, +0.98 ± 0.54; RdRP/S genes, +2.64 ± 0.48. On the other hand, the number of tests performed within 24 h raised from 86.4% to 97.8%, the turn-around time decreased from 19:18 to 09:03 (p < .0001), and the number of tests that can be performed per day doubled since this technique was introduced routinely in our laboratory.


Assuntos
/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , /isolamento & purificação , /diagnóstico , /genética , Surtos de Doenças , Europa (Continente) , Genes Virais , Humanos , Fosfoproteínas/genética , RNA Viral/análise , RNA Viral/genética , Sensibilidade e Especificidade
15.
Biosens Bioelectron ; 178: 113015, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33493896

RESUMO

Dependable, specific and rapid diagnostic methods for severe acute respiratory syndrome ß-coronavirus (SARS-CoV-2) detection are needed to promote public health interventions for coronavirus disease 2019 (COVID-19). Herein, we have established an entropy-driven amplified electrochemiluminescence (ECL) strategy to detect the RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2 known as RdRp-COVID which as the target for SARS-CoV-2 plays an essential role in the diagnosis of COVID-19. For the construction of the sensors, DNA tetrahedron (DT) is modified on the surface of the electrode to furnish robust and programmable scaffolds materials, upon which target DNA-participated entropy-driven amplified reaction is efficiently conducted to link the Ru (bpy)32+ modified S3 to the linear ssDNA at the vertex of the tetrahedron and eventually present an "ECL on" state. The rigid tetrahedral structure of the DT probe enhances the ECL intensity and avoids the cross-reactivity between single-stranded DNA, thus increasing the sensitivity of the assays. The enzyme-free entropy-driven reaction prevents the use of expensive enzyme reagents and facilitates the realization of large-scale screening of SARS-CoV-2 patients. Our DT-based ECL sensor has demonstrated significant specificity and high sensitivity for SARS-CoV-2 with a limit of detection (LOD) down to 2.67 fM. Additionally, our operational method has achieved the detection of RdRp-COVID in human serum samples, which supplies a reliable and feasible sensing platform for the clinical bioanalysis.


Assuntos
Técnicas Biossensoriais/instrumentação , /diagnóstico , /genética , /isolamento & purificação , Técnicas Biossensoriais/estatística & dados numéricos , /sangue , DNA/química , Técnicas Eletroquímicas , Entropia , Genes Virais , Humanos , Limite de Detecção , Luminescência , Conformação de Ácido Nucleico , Pandemias , Sensibilidade e Especificidade
16.
Sci Rep ; 11(1): 2261, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33500503

RESUMO

The diagnosis of COVID-19 relies on the direct detection of SARS-CoV-2 RNA in respiratory specimens by RT-PCR. The pandemic spread of the disease caused an imbalance between demand and supply of materials and reagents needed for diagnostic purposes including swab sets. In a comparative effectiveness study, we conducted serial follow-up swabs in hospitalized laboratory-confirmed COVID-19 patients. We assessed the diagnostic performance of an in-house system developed according to recommendations by the US CDC. In a total of 96 serial swabs, we found significant differences in the accuracy of the different swab systems to generate a positive result in SARS-CoV-2 RT-PCR, ranging from around 50 to 80%. Of note, an in-house swab system was superior to most commercially available sets as reflected by significantly lower Ct values of viral genes. Thus, a simple combination of broadly available materials may enable diagnostic laboratories to bypass global limitations in the supply of swab sets.


Assuntos
/instrumentação , Equipamentos Descartáveis/provisão & distribução , Técnicas de Diagnóstico Molecular/instrumentação , /isolamento & purificação , /métodos , Técnicas de Laboratório Clínico , Testes Diagnósticos de Rotina , Genes Virais , Humanos , Técnicas de Diagnóstico Molecular/métodos , Controle de Qualidade , RNA Viral/análise , Reprodutibilidade dos Testes , Alocação de Recursos , Manejo de Espécimes
17.
J Infect Public Health ; 14(2): 238-243, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33493920

RESUMO

BACKGROUND: The MERS-CoV was identified for the first time from Jeddah, Saudi Arabia in 2012 from a hospitalized patient. This virus has now been spread to 27 countries with a total of 858 deaths and 2494 confirmed cases and has become a serious concern for the human population. Camels are well known for the transmission of the virus to the human population. METHODS: In this report, we have discussed the designing, prediction, and evaluation of potential siRNAs against the orf1ab gene of MERS-CoV. The online software was used to predict and design the siRNAs and finally, total twenty-one siRNA were filtered out from four hundred and sixty-two sIRNAs as per their scoring and specificity criteria. We have used only ten siRNAs to evaluate their cytotoxicity and efficacy by reverse transfection approach in HEK-293-T cell lines. RESULTS: Based on the results and data generated; no cytotoxicity was observed for any siRNAs at various concentrations in HEK-293-T cells. The ct value of real-time PCR showed the inhibition of viral replication in siRNA-1, 2, 4, 6, and 9. The data generated provided the preliminary information and encouraged us to evaluate the remaining siRNAs separately as well as in combination to analyses the replication of MERS-CoV inhibition in other cell lines. CONCLUSION: Based on the results obtained; it is concluded that the prediction of siRNAs using online software resulted in the filtration of potential siRNAs with high accuracy and strength. This technology can be used to design and develop antiviral therapy not only for MERS-CoV but also against other viruses.


Assuntos
Genes Virais , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , RNA Interferente Pequeno/genética , Animais , Camelus , Infecções por Coronavirus , Células HEK293 , Humanos , Software
18.
Viruses ; 13(1)2021 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-33435393

RESUMO

The bacteriophage T4 early gene product MotB binds tightly but nonspecifically to DNA, copurifies with the host Nucleoid Associated Protein (NAP) H-NS in the presence of DNA and improves T4 fitness. However, the T4 transcriptome is not significantly affected by a motB knockdown. Here we have investigated the phylogeny of MotB and its predicted domains, how MotB and H-NS together interact with DNA, and how heterologous overexpression of motB impacts host gene expression. We find that motB is highly conserved among Tevenvirinae. Although the MotB sequence has no homology to proteins of known function, predicted structure homology searches suggest that MotB is composed of an N-terminal Kyprides-Onzonis-Woese (KOW) motif and a C-terminal DNA-binding domain of oligonucleotide/oligosaccharide (OB)-fold; either of which could provide MotB's ability to bind DNA. DNase I footprinting demonstrates that MotB dramatically alters the interaction of H-NS with DNA in vitro. RNA-seq analyses indicate that expression of plasmid-borne motB up-regulates 75 host genes; no host genes are down-regulated. Approximately 1/3 of the up-regulated genes have previously been shown to be part of the H-NS regulon. Our results indicate that MotB provides a conserved function for Tevenvirinae and suggest a model in which MotB functions to alter the host transcriptome, possibly by changing the association of H-NS with the host DNA, which then leads to conditions that are more favorable for infection.


Assuntos
Bactérias/metabolismo , Bactérias/virologia , Proteínas de Bactérias/metabolismo , Bacteriófago T4/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Genes Virais , Interações Hospedeiro-Patógeno , Proteínas de Bactérias/química , Sequência de Bases , Proteínas de Ligação a DNA/química , Filogenia , Fagos T/genética
19.
Biomol NMR Assign ; 15(1): 203-211, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33484403

RESUMO

The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain cis-acting RNA elements that affect RNA translation and replication. The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5'-untranslated region (5'-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5'-UUUCGU-3' hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive 1H, 13C and 15N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy.


Assuntos
Regiões 5' não Traduzidas , Espectroscopia de Ressonância Magnética , /genética , Isótopos de Carbono , Genes Virais , Hidrogênio , Isótopos de Nitrogênio , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína
20.
Nat Commun ; 12(1): 27, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397904

RESUMO

Bacteriophages (phages), or bacterial viruses, are very diverse and highly abundant worldwide, including as a part of the human microbiomes. Although a few metagenomic studies have focused on oral phages, they relied on short-read sequencing. Here, we conduct a long-read metagenomic study of human saliva using PromethION. Our analyses, which integrate both PromethION and HiSeq data of >30 Gb per sample with low human DNA contamination, identify hundreds of viral contigs; 0-43.8% and 12.5-56.3% of the confidently predicted phages and prophages, respectively, do not cluster with those reported previously. Our analyses demonstrate enhanced scaffolding, and the ability to place a prophage in its host genomic context and enable its taxonomic classification. Our analyses also identify a Streptococcus phage/prophage group and nine jumbo phages/prophages. 86% of the phage/prophage group and 67% of the jumbo phages/prophages contain remote homologs of antimicrobial resistance genes. Pan-genome analysis of the phages/prophages reveals remarkable diversity, identifying 0.3% and 86.4% of the genes as core and singletons, respectively. Furthermore, our study suggests that oral phages present in human saliva are under selective pressure to escape CRISPR immunity. Our study demonstrates the power of long-read metagenomics utilizing PromethION in uncovering bacteriophages and their interaction with host bacteria.


Assuntos
Bactérias/virologia , Bacteriófagos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/genética , Metagenômica , Boca/microbiologia , Boca/virologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Contaminação por DNA , DNA Viral/genética , Resistência Microbiana a Medicamentos/genética , Genes Virais , Genoma Bacteriano , Humanos , Integrases/genética , Metagenoma , Prófagos/genética , Proteômica , Streptococcus/virologia
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