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1.
Article in Chinese | WPRIM | ID: wpr-1046011

ABSTRACT

To explore the biological characteristics related to the pathogenesis and severity of respiratory syncytial virus (RSV) bronchiolitis by RNA sequencing of white blood cells in children with RSV bronchiolitis. This study is a case-control study. A total of 87 children diagnosed with bronchiolitis and RSV antigen positive and/or RSV nucleic acid positive in the pediatric respiratory department of the Second Affiliated Hospital of Wenzhou Medical University from October 2019 to April 2022 were selected as the case group. The case group was divided into three groups based on the condition: mild, moderate, and severe, and there were two groups according to the presence or absence of atopic symptoms: the atopic group and the non-atopic group, forty healthy children in the same period were selected as the control group. The whole blood leukocyte RNA of the children in the case group and the control group was extracted for RNA sequencing, and the data were analyzed to obtain differentially expressed genes (DEGs). Then, the immunobiological pathways and genes related to the pathogenesis, disease condition, and atopy were screened through Gene Ontology (GO) annotation, Kyoto Gene and Genome Encyclopedia (KEGG) annotation, and protein interaction network (PPI) construction methods. Construct the weighted gene co-expression network analysis (WGCNA) module to identify potential biological indicators related to disease severity.Compared with the control group, the case group had a total of 1 782 DEGs, including 1 586 upregulated genes and 196 downregulated genes. The GO pathway enrichment of DEGs is mainly enriched in molecular functions such as peroxidase activity and oxidoreductase activity. In the cytological components, it is mainly enriched in cytoplasmic vesicle lumen and secretory granule lumen. In biological processes, it is mainly enriched in processes such as neutrophil activation involved in immune responses, neutrophil degranulation, and neutrophil activation. KEGG analysis is mainly concentrated in the signal pathway of the viral protein interaction with cytokine and cytokine receptor. A PPI network was constructed to screen four genes at the core position, including CCL2, IL-10, MMP9 and JUN. The DEGs obtained by comparing different disease groups with the control group are mainly enriched in retrograde endocannabinoid signaling and cell apoptosis pathways. WGCNA analysis showed that the brown module related to oxygen saturation was most closely related to the disease, and its gene was mainly enriched in the RNA helicase retinoic acid inducible gene-I (RIG-I) like receptor signal pathway. There are 230 specific DEGs in the atopic group and 444 in the non-atopic group. KEGG enrichment analysis results show that both groups are enriched to NF-κB signaling pathway, the characteristic does not cause significant changes in immune response and transcriptome characteristics in children with RSV bronchiolitis. In conclusion, neutrophil activation, degranulation pathway and signal pathway of interaction between viral protein and cytokine and cytokine receptor are involved in the immune response of RSV bronchiolitis host. CCL2, IL-10, MMP9 and JUN genes may be associated with the pathogenesis. They might be potential biomarkers related to disease severity in RIG-I like receptors, cell apoptosis, and endogenous cannabinoid related signaling pathways.


Subject(s)
Child , Humans , Transcriptome , Interleukin-10 , Matrix Metalloproteinase 9 , Case-Control Studies , Sequence Analysis, RNA , Respiratory Syncytial Virus Infections/genetics , Receptors, Cytokine , Viral Proteins , Respiratory Syncytial Viruses , Computational Biology/methods
2.
Article in Chinese | WPRIM | ID: wpr-1046334

ABSTRACT

To explore the biological characteristics related to the pathogenesis and severity of respiratory syncytial virus (RSV) bronchiolitis by RNA sequencing of white blood cells in children with RSV bronchiolitis. This study is a case-control study. A total of 87 children diagnosed with bronchiolitis and RSV antigen positive and/or RSV nucleic acid positive in the pediatric respiratory department of the Second Affiliated Hospital of Wenzhou Medical University from October 2019 to April 2022 were selected as the case group. The case group was divided into three groups based on the condition: mild, moderate, and severe, and there were two groups according to the presence or absence of atopic symptoms: the atopic group and the non-atopic group, forty healthy children in the same period were selected as the control group. The whole blood leukocyte RNA of the children in the case group and the control group was extracted for RNA sequencing, and the data were analyzed to obtain differentially expressed genes (DEGs). Then, the immunobiological pathways and genes related to the pathogenesis, disease condition, and atopy were screened through Gene Ontology (GO) annotation, Kyoto Gene and Genome Encyclopedia (KEGG) annotation, and protein interaction network (PPI) construction methods. Construct the weighted gene co-expression network analysis (WGCNA) module to identify potential biological indicators related to disease severity.Compared with the control group, the case group had a total of 1 782 DEGs, including 1 586 upregulated genes and 196 downregulated genes. The GO pathway enrichment of DEGs is mainly enriched in molecular functions such as peroxidase activity and oxidoreductase activity. In the cytological components, it is mainly enriched in cytoplasmic vesicle lumen and secretory granule lumen. In biological processes, it is mainly enriched in processes such as neutrophil activation involved in immune responses, neutrophil degranulation, and neutrophil activation. KEGG analysis is mainly concentrated in the signal pathway of the viral protein interaction with cytokine and cytokine receptor. A PPI network was constructed to screen four genes at the core position, including CCL2, IL-10, MMP9 and JUN. The DEGs obtained by comparing different disease groups with the control group are mainly enriched in retrograde endocannabinoid signaling and cell apoptosis pathways. WGCNA analysis showed that the brown module related to oxygen saturation was most closely related to the disease, and its gene was mainly enriched in the RNA helicase retinoic acid inducible gene-I (RIG-I) like receptor signal pathway. There are 230 specific DEGs in the atopic group and 444 in the non-atopic group. KEGG enrichment analysis results show that both groups are enriched to NF-κB signaling pathway, the characteristic does not cause significant changes in immune response and transcriptome characteristics in children with RSV bronchiolitis. In conclusion, neutrophil activation, degranulation pathway and signal pathway of interaction between viral protein and cytokine and cytokine receptor are involved in the immune response of RSV bronchiolitis host. CCL2, IL-10, MMP9 and JUN genes may be associated with the pathogenesis. They might be potential biomarkers related to disease severity in RIG-I like receptors, cell apoptosis, and endogenous cannabinoid related signaling pathways.


Subject(s)
Child , Humans , Transcriptome , Interleukin-10 , Matrix Metalloproteinase 9 , Case-Control Studies , Sequence Analysis, RNA , Respiratory Syncytial Virus Infections/genetics , Receptors, Cytokine , Viral Proteins , Respiratory Syncytial Viruses , Computational Biology/methods
3.
Chinese Journal of Biotechnology ; (12): 4849-4860, 2023.
Article in Chinese | WPRIM | ID: wpr-1008063

ABSTRACT

Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.


Subject(s)
Animals , Foot-and-Mouth Disease Virus/genetics , Capsid Proteins , Viral Proteins/metabolism , Foot-and-Mouth Disease/prevention & control , Tetracyclines/metabolism , Viral Vaccines , Antibodies, Viral , Mammals/metabolism
4.
Biomed. environ. sci ; Biomed. environ. sci;(12): 133-140, 2022.
Article in English | WPRIM | ID: wpr-927643

ABSTRACT

OBJECTIVE@#To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene.@*METHODS@#A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay.@*RESULTS@#The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses.@*CONCLUSION@#A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.


Subject(s)
Animals , African Swine Fever/virology , African Swine Fever Virus/isolation & purification , Nucleic Acid Amplification Techniques/methods , Recombinases/chemistry , Sensitivity and Specificity , Swine , Viral Proteins/genetics
5.
Chinese Journal of Biotechnology ; (12): 1824-1836, 2022.
Article in Chinese | WPRIM | ID: wpr-927820

ABSTRACT

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×109.1 TCID50/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.


Subject(s)
Animals , Humans , Mice , Adenoviridae/genetics , Adenoviruses, Human/genetics , Antibodies, Viral , Capsid/metabolism , Capsid Proteins , Foot-and-Mouth Disease/prevention & control , Foot-and-Mouth Disease Virus/genetics , HEK293 Cells , Recombinant Proteins/genetics , Serogroup , Swine , Viral Proteins , Viral Vaccines/genetics
6.
Zhonghua Yu Fang Yi Xue Za Zhi ; (12): 626-631, 2022.
Article in Chinese | WPRIM | ID: wpr-935335

ABSTRACT

The purpose was to discuss the infection status of human parainfluenza virus type 3 (HPIV-3) in children with acute respiratory tract infection(ARTI) in Qingdao, Shandong province, and to analyze the gene characteristics of HPIV-3 hemagglutinin-neuraminidase protein (HN). This study was a cross-sectional study. A total of 1 674 throat swab samples were collected randomly from children with ARTI, in the three hospitals (Qingdao Women and Children's Hospital, West Coast Branch of Affiliated Hospital of Qingdao University, Laoshan Branch of Affiliated Hospital of Qingdao University) from January 2018 to December 2019. Multiplex real-time fluorescence RT-PCR was performed to screen HPIV-3 positive specimens. For HPIV-3 positive specimens, nested PCR was used to amplify the full-length HN gene of HPIV-3. The HN gene was sequenced and compared with the representative strains of HPIV-3 in GenBank, and the phylogenetic tree was established. As results, this study collected 1 674 samples, in which there were 90 HPIV-3 positive samples showed and the detection rate was 5.37%. Among positive specimens, the number of samples from children under 6 years old was 88, accounting for 97.78%. HPIV-3 positive cases were mainly distributed in spring and summer. The full-length sequences of 44 HPIV-3 HN genes were obtained by nested PCR method. Sequence alignment and evolutionary analysis showed that the HPIV-3HN gene belonged to the C3a and C3b branches of C3 genotype, with 30 strains of subtype C3a and 14 strains of subtype C3b. The nucleotide and amino acid homology of the amplified 44 strains of the HPIV-3 HN gene in Qingdao were 97.0%-100.0% and 98.5%-100.0%, respectively. In conclusion, from 2018 to 2019, the C3a and C3b branches of HPIV-3 C3 genotype were circulating prevalent in Qingdao, Shandong province. HN gene variation rate was low, but showed certain regional characteristics in evolution.


Subject(s)
Child , Child, Preschool , Female , Humans , Cross-Sectional Studies , Hemagglutinins , Neuraminidase , Parainfluenza Virus 3, Human/genetics , Phylogeny , Respiratory Tract Infections/epidemiology , Viral Proteins
7.
Rev. ADM ; 78(5): 275-279, sept.-oct. 2021.
Article in Spanish | LILACS | ID: biblio-1348224

ABSTRACT

El SARS-CoV-2, causante de que estemos viviendo una pandemia mundial, tuvo sus orígenes en China, desde donde ha traspasado fronteras rápidamente, llegando a todos los rincones del mundo. Muchos han sido los equipos de investigación que se enfrentan el reto de conseguir una vacuna que logre combatir este mortal virus. Es por este motivo que en esta investigación se pretendió analizar la bibliografía referida a la vacuna Johnson & Johnson (J&J) contra COVID-19: distribución mundial de la vacuna, mecanismo de acción, indicaciones, contraindicaciones y efectos secundarios. Varios estudios demuestran que su eficacia varía de acuerdo con la edad y género de cada individuo; sin embargo, esta vacuna alcanzó un grado de certeza moderada. Los efectos adversos en su mayoría son leves y se resolvieron al cabo de dos días, siendo excepción algunos casos, ya que se registró un efecto adverso poco común denominado trombocitopenia prevalente en mujeres de 18 a 40 años, por este motivo, la FDA (Administración de Alimentos y Medicamentos de EE.UU.) recomienda la precaución en el uso de la vacuna con respecto a este efecto adverso que en algunos casos podría ser mortal (AU)


The SARS-CoV-2, which caused us to be experiencing a global pandemic, had its origins in China, from where it has crossed borders rapidly, reaching all corners of the world. Many research teams have faced the challenge of getting a vaccine to fight this deadly virus. For this reason, this research aimed to analyze the literature on the Johnson & Johnson COVID-19 vaccine: global distribution of the vaccine, mechanism of action, indications, contraindications and side effects. Several studies show that its effectiveness varies according to the age and gender of each individual, but this vaccine reached a moderate degree of certainty. The adverse effects are mostly mild and resolved within two days, with some exceptions being a rare adverse effect called prevalent thrombocytopenia in women aged 18 to 40 years. For this reason, the FDA recommends caution in the use of the vaccine with respect to this potentially fatal adverse effect in some cases (AU)


Subject(s)
Humans , Male , Female , Contraindications, Drug , COVID-19 Vaccines/adverse effects , COVID-19 Vaccines/supply & distribution , COVID-19 Vaccines/therapeutic use , COVID-19 Vaccines/pharmacology , SARS-CoV-2 , COVID-19/prevention & control , United States Food and Drug Administration , Viral Proteins , Effectiveness , RNA, Viral , Sex Factors , Age Factors , Virus Inactivation
8.
Med. UIS ; 34(2): 61-75, mayo-ago. 2021. tab, graf
Article in Spanish | LILACS | ID: biblio-1375820

ABSTRACT

RESUMEN La enfermedad por coronavirus 2019 (COVID-19) es causada por un nuevo betacoronavirus conocido como síndrome respiratorio agudo severo coronavirus-2 (SARS-CoV-2). Para el 22 de junio del 2021, el número de casos confirmados en todo el mundo había superado los 178 millones, con más de 3 millones de muertes. La fisiopatología de la COVID-19 a partir de la infección por SARS-CoV-2 no está del todo dilucidada. En el presente artículo se exponen los hallazgos encontrados después de la búsqueda en la literatura científica realizada en la base de datos PubMed entre octubre de 2020 y abril de 2021 en la cual se incluyeron 71 artículos, con el objetivo de la revisión fisiopatológica completa, detallada y actualizada del SARS-CoV-2, abordando temas como la caracterización y ciclo de vida del virus, el mecanismo de transmisión, la cinética viral y la respuesta inmune, junto con la dinámica fisiopatológica de la infección. MÉD.UIS.2021;34(2): 61-75.


ABSTRACT Coronavirus disease 2019 (COVID-19) is caused by a new betacoronavirus named as Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). On June 22nd, 2021, the number of confirmed cases worldwide exceeded 178 million, resulting in more than 3 million deaths. The pathophysiology of COVID-19 from the infection of SARS-CoV-2 is not entirely elucidated. This review presents the findings after the research in the scientific literature carried out in the PubMed database between October 2020 and April 2021, in which 71 articles were included, with the aim of a complete, detailed and updated pathophysiological review of SARS-CoV-2, addressing issues such as the characterization and life cycle of the virus, the transmission mechanism, viral kinetics and immune response, along with the pathophysiological dynamics of the infection. MÉD.UIS.2021;34(2): 61-75.


Subject(s)
Humans , COVID-19 , Viral Proteins , SARS-CoV-2 , Immunity
9.
Medicina (B.Aires) ; Medicina (B.Aires);80(2): 162-168, abr. 2020. ilus, graf
Article in Spanish | LILACS | ID: biblio-1125058

ABSTRACT

En abril de 2019, UNICEF denunció que más de 20 millones de niños en todo el mundo no habían sido vacunados y alertó sobre posibles brotes de sarampión que, por su alta contagiosidad, es la primera enfermedad en emerger entre las prevenibles mediante vacunación. De continuar el descenso en las vacunaciones, podrían reaparecer también pertussis, tétanos y otras enfermedades con menor requerimiento de cobertura para alcanzar protección poblacional. A fin de agosto de 2019 se inició en la Argentina el actual brote de sarampión. Este virus se transmite por vía respiratoria, infecta múltiples órganos e induce inmunosupresión. Su genoma consiste en ARN de cadena simple. La genotipificación se efectúa por secuenciación de un fragmento de 450 nucleótidos de la proteína N que contiene la mayor densidad de variación de nucleótidos del genoma. En Sudamérica circula el genotipo D8, y en Norteamérica hay, además, un 8% de genotipo B3. Cada persona con sarampión infecta, en promedio, otras 12-18 en una población susceptible. La vacunación confiere protección directa e indirecta, e induce tanto anticuerpos como inmunidad celular. Los recién nacidos tienen protección hasta los 6 meses por anticuerpos maternos transmitidos vía placentaria. En la Argentina, el Calendario de Vacunación incluye dos dosis de triple viral, a los 12 meses y a los 5 años, y una dosis cero (6- 11 meses de edad) en distritos con casos de enfermedad. Una dosis protege al 93% de los vacunados a los 12 meses y dos dosis al 97%, de por vida.


In April 2019, UNICEF denounced that more than 20 million children worldwide had not been vaccinated and alerted on possible outbreaks of measles which, due to the high transmissibility of this virus, is the first disease preventable by vaccination to emerge. If the decline in vaccinations continues, pertussis, tetanus and other diseases, which require less coverage to achieve population protection, may also reappear. In Argentina, the current outbreak began in late August 2019. Measles virus is transmitted by air, infects multiple organs, and is associated with immunosuppression. Its genome consists of single stranded RNA. Genotyping is carried out by sequencing a 450-nucleotide fragment of the N protein, which contains the highest density of nucleotide variation. In South America, D8 is the circulating genotype and in North America, B3 accounts for 8% of the cases. Each person with measles infects, on average, another 12-18 people in a susceptible population. Vaccination confers direct and indirect protection, and induces both antibodies and cellular immunity. Newborns are protected by maternal antibodies transmitted via the placenta, up to 6 months. In Argentina, the Vaccination Calendar includes two doses of triple viral vaccine, at 12 months and 5 years, and a zero dose (6- 11 months of age) in districts with disease cases. The protection conferred by the vaccine is 93% at 12 months with a dose, and with 2 doses 97% for life.


Subject(s)
Humans , Infant , Child, Preschool , History, 19th Century , Vaccination , Measles/prevention & control , Argentina/epidemiology , Viral Proteins , Disease Outbreaks , Nucleocapsid Proteins , Genotype , Measles/history , Measles/epidemiology , Measles/virology , Nucleoproteins
10.
Arq. neuropsiquiatr ; Arq. neuropsiquiatr;78(3): 163-168, Mar. 2020. tab
Article in English | LILACS | ID: biblio-1098075

ABSTRACT

Abstract Herpes simplex virus (HSV) is a cause of a severe disease of the central nervous system (CNS) in humans. The demonstration of specific antibodies in the cerebrospinal fluid (CSF) may contribute to the retrospective neurological diagnosis. However, the commercial immunological tests for HSV infection are for use in serum samples. Objective: The aim of the present study was to adapt a commercial kit anti-HSV IgG used for serum samples to be performed with a CSF sample. Methods: Forty CSF specimens from 38 patients with suspected CNS HSV infection were serially diluted for detecting anti-HSV IgG by enzyme immunoassay (EIA). The same samples were also analyzed with the polymerase chain reaction (PCR). Results: The sensitivity of EIA test for HSV was 5% (dilution 1:40) and 65% (dilution 1:2) in CSF, and HSV DNA PCR was 15%. The combined analysis of EIA (dilution 1:2) and PCR increased the sensitivity up to 72.5%. The inflammatory CSF was associated with positive HSV PCR. Conclusions: We demonstrated the importance to adapt serological anti-HSV IgG EIA test for CSF assays to increase the accuracy of the analysis, considering the low concentration of specific antibodies in CSF.


Resumo O vírus herpes simples (HSV) é um dos agentes causadores de uma doença grave no sistema nervoso central (SNC) em humanos. A detecção de anticorpos específicos no líquido cefalorraquidiano (LCR) pode contribuir para o diagnóstico neurológico retrospectivo. Entretanto, os testes imunológicos comerciais são para uso em amostras de soro. Objetivo: Adaptar um kit comercial sorológico anti-HSV IgG para ser utilizado no de LCR. Metodos: Quarenta amostras de LCR de 38 pacientes com suspeita de infecção por HSV no SNC foram diluídas pesquisa de anticorpos anti-HSV IgG pelo método imunoenzimático (EIA). Além disso, as mesmas amostras também foram analisadas por reação em cadeia da polimerase (PCR). Resultados: A sensibilidade do teste EIA para o HSV consistiu em 5% (diluição 1:40) e 65% (diluição 1:2) no LCR, e o PCR do DNA do HSV, 15%. A análise combinada de EIA (diluição 1:2) e PCR aumentou a sensibilidade para 72,5%. Houve associação entre presença do LCR inflamatório e PCR positiva para HSV. Conclusões: Demonstramos a importância na adaptação previa do teste sorológico anti-HSV IgG EIA para ensaios do no LCR, a fim de aumentar a acuracia da análise, considerando a baixa concentração de anticorpos específicos no LCR.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Cerebrospinal Fluid/virology , Simplexvirus/isolation & purification , Herpes Simplex/diagnosis , Herpes Simplex/virology , Antibodies, Viral/cerebrospinal fluid , Viral Proteins , DNA, Viral/genetics , Polymerase Chain Reaction/methods , Retrospective Studies , Simplexvirus/genetics , DNA-Directed DNA Polymerase/genetics , Exodeoxyribonucleases , Herpes Simplex/cerebrospinal fluid , Nervous System
11.
Chinese Journal of Biotechnology ; (12): 2467-2477, 2020.
Article in Chinese | WPRIM | ID: wpr-878503

ABSTRACT

The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.


Subject(s)
Anabaena/genetics , Cloning, Molecular , DNA-Directed RNA Polymerases , Escherichia coli/genetics , Gene Expression , Mercury , Plasmids , Viral Proteins
12.
Frontiers of Medicine ; (4): 746-751, 2020.
Article in English | WPRIM | ID: wpr-880959

ABSTRACT

The ongoing pandemic of Coronavirus disease 19 (COVID-19) is caused by a newly discovered β Coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). How long the adaptive immunity triggered by SARS-CoV-2 can last is of critical clinical relevance in assessing the probability of second infection and efficacy of vaccination. Here we examined, using ELISA, the IgG antibodies in serum specimens collected from 17 COVID-19 patients at 6-7 months after diagnosis and the results were compared to those from cases investigated 2 weeks to 2 months post-infection. All samples were positive for IgGs against the S- and N-proteins of SARS-CoV-2. Notably, 14 samples available at 6-7 months post-infection all showed significant neutralizing activities in a pseudovirus assay, with no difference in blocking the cell-entry of the 614D and 614G variants of SARS-CoV-2. Furthermore, in 10 blood samples from cases at 6-7 months post-infection used for memory T-cell tests, we found that interferon γ-producing CD4


Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Adaptive Immunity/physiology , Antibodies, Neutralizing/blood , COVID-19/immunology , Cohort Studies , Immunoglobulin G/blood , SARS-CoV-2/immunology , T-Lymphocytes/physiology , Time Factors , Viral Proteins/immunology
13.
Chinese Journal of Biotechnology ; (12): 1113-1125, 2020.
Article in Chinese | WPRIM | ID: wpr-826866

ABSTRACT

ORF3 protein, the single accessory protein encoded by porcine epidemic diarrhea virus (PEDV), is related to viral pathogenicity. In order to determine the cytoplasmic location signal of PEDV ORF3, we constructed a series of recombinant plasmids carrying full-length or truncated segments of PEDV DR13 ORF3 protein. When the acquired plasmids were transfected into Vero cells, expression and distribution of the EGFP-fused full-length ORF3 protein and its truncated forms in the cells were observed by laser confocal microscopy. The results showed that ORF3 protein or their truncated forms containing 40-91 aa segment including two transmembrane domains were localized in the cytoplasm, whereas ORF3 truncated peptides without the 40-91 aa segment were distributed in the whole cell (in both cytoplasm and nucleus). This suggests that the 40-91 aa is the key structural domain determining cytoplasmic location of PEDV ORF3 protein. The discovery provides reference for further clarifying intracellular transport and biological function of PEDV ORF3 protein.


Subject(s)
Animals , Amino Acid Sequence , Chlorocebus aethiops , Coronavirus Infections , Virology , Cytoplasm , Virology , Porcine epidemic diarrhea virus , Genetics , Protein Domains , Swine , Vero Cells , Viral Proteins , Chemistry , Metabolism
14.
Protein & Cell ; (12): 894-914, 2020.
Article in English | WPRIM | ID: wpr-880885

ABSTRACT

Tripartite motif (TRIM) family proteins are important effectors of innate immunity against viral infections. Here we identified TRIM35 as a regulator of TRAF3 activation. Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon (IFN) in response to viral infection. Trim35-deficient mice were more susceptible to influenza A virus (IAV) infection than were wild-type mice. TRIM35 promoted the RIG-I-mediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1. IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3. TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2, thereby antagonizing its suppression of TRAF3 activation. Our in vitro and in vivo findings thus reveal novel roles of TRIM35, through catalyzing Lys63- or Lys48-linked polyubiquitination, in RIG-I antiviral immunity and mechanism of defense against IAV infection.


Subject(s)
Animals , Dogs , Humans , Mice , A549 Cells , Apoptosis Regulatory Proteins/immunology , DEAD Box Protein 58/immunology , HEK293 Cells , Influenza A Virus, H1N1 Subtype/immunology , Madin Darby Canine Kidney Cells , Mice, Knockout , Orthomyxoviridae Infections/pathology , Proteolysis , Signal Transduction/immunology , THP-1 Cells , TNF Receptor-Associated Factor 3/immunology , Ubiquitination/immunology , Viral Proteins/immunology
15.
Rev. peru. med. exp. salud publica ; 36(1): 46-53, ene.-mar. 2019. tab, graf
Article in Spanish | LILACS | ID: biblio-1004412

ABSTRACT

RESUMEN Objetivos. Caracterizar la nucleoproteína (N) y establecer el origen del virus de la rabia en canes procedentes de Arequipa. Materiales y métodos. Se analizaron 30 muestras de tejido nervioso procedentes de los departamentos de Arequipa y Puno. Se extrajo el ARN total de las muestras y se sintetizó ADNc para amplificar el gen de la nucleoproteína, secuenciarlo y realizar el análisis bioinformático. Resultados. Se obtuvo la formación de un grupo definido con respecto al grupo externo (European bat lyssavirus). Este grupo fue clasificado en dos subgrupos, uno constituido por muestras procedentes de Puno y Arequipa (subgrupo A), y otro por muestras de Puno (subgrupo B), observándose una identidad nucleotídica de 99,9% en el subgrupo A. Conclusiones. Los agrupamientos de las secuencias virales muestran que los casos de rabia canina notificados en Arequipa son el resultado de la expansión de rabia canina procedente de la región endémica de Puno.


ABSTRACT Objective . To characterize the nucleoprotein (N) and establish the origin of the rabies virus in dogs coming from Arequipa. Materials and Methods. Thirty samples of nervous tissue from the departments of Arequipa and Puno were analyzed. Total RNA was extracted from the samples and cDNA was synthesized to amplify the nucleoprotein gene, sequence it, and perform bioinformatics analysis. Results . A defined group was formed with respect to the external group (European bat lyssavirus). This group was classified into two subgroups, one constituted by samples coming from Puno and Arequipa (subgroup A), and another one by samples from Puno (subgroup B), exhibiting a nucleotide identity of 99.9% in subgroup A. This group was classified in two subgroups, one constituted by samples coming from Puno and Arequipa (subgroup A), and another one by samples from Puno (subgroup B), observing a nucleotide identity of 99.9% in subgroup A. Conclusions. The groupings of viral sequences show that the cases of canine rabies reported in Arequipa are the result of the expansion of canine rabies from the endemic region of Puno.


Subject(s)
Animals , Dogs , Rabies virus/genetics , Viral Proteins/genetics , Nucleoproteins/genetics , Peru
16.
Article in Chinese | WPRIM | ID: wpr-771402

ABSTRACT

A novel protein encoded by the open reading frame 4 (ORF4) was recently discovered in porcine circovirus type 2 (PCV2). However, little is known about the interaction proteins of ORF4 which hindered better understanding the biological functions of ORF4 in the life cycle of PCV2. In the present study, the ORF4 was inserted into the multiple cloning site of pCMV-N-Flag-GST, yielding recombinant plasmid pCMV-N-Flag-GST-ORF4. The recombinant plasmid was transfected into 293T cells and the intracellular interaction complex of ORF4 were enriched and separated by GST pull-down and SDS-PAGE, sequentially. The potential interacting proteins of PCV2 ORF4 were stained with silver and identified by mass spectrometry (MS). Finally, five candidate ORF4-interacting proteins, including Serine/threonine-protein phosphatase 6 catalytic subunit, alpha cardiac muscle 1, actin, SEC14-like protein 5 and myosin 9 were identified. These results would benefit a better understanding of the biological function of ORF4 in PCV2 infected cells.


Subject(s)
Animals , Humans , Circoviridae Infections , Circovirus , HEK293 Cells , Mass Spectrometry , Open Reading Frames , Swine , Viral Proteins
17.
Bol. micol. (Valparaiso En linea) ; 33(2): 10-16, dic. 2018. tab, ilus
Article in Spanish | LILACS | ID: biblio-1008155

ABSTRACT

Los virus han acompañado al hombre a través de toda su historia, provocando infecciones y plagas que han generado la muerte y el temor de poblaciones completas. Muchos han sido los investigadores que han contribuido a conocer la estructura, patogenia e inmunidad involucrada. Es fundamental conocer los aspectos básicos de los virus para entender la enfermedad y el tratamiento, por lo que esperamos contribuir a esto con los apuntes que a continuación presentamos...(AU)


Viruses have accompanied humankind through their history, causing infections and producing death and fear in population. A lot of researchers have contributed to know the structure, pathogeny and immunity involved. To understand the disease and treatment is fundamental to know the basic aspects, which is the reason to present these notes...(AU)


Subject(s)
Viruses/pathogenicity , Nucleic Acids , Viral Proteins , Virology , Viruses/classification , Viruses/ultrastructure
18.
Rev. Univ. Ind. Santander, Salud ; 50(3): 257-268, Julio 23, 2018. tab, graf
Article in Spanish | LILACS | ID: biblio-957518

ABSTRACT

Resumen La infección crónica con virus oncogénicos es responsable de aproximadamente el 20% de todos los cánceres reportados en humanos, este proceso de oncogénesis viral presenta una naturaleza compleja, multietapa y multifactorial. Un ejemplo de ello es el Virus de Epstein- Barr (EBV), un herpesvirus que infecta de manera latente a más del 90% de la población. Aunque la infección a menudo cursa de manera asintomática, el EBV es capaz de modificar su expresión genómica estableciendo diferentes fases de latencia, alterando así el metabolismo de sus células blanco, como son los linfocitos B y las células epiteliales, proceso que resulta determinante en la aparición y desarrollo de diferentes patologías que van desde la mononucleosis infecciosa hasta procesos oncológicos como el linfoma de Burkitt, el cáncer gástrico o el cáncer nasofaríngeo.


Abstract Chronic infection with oncogenic viruses is responsible for approximately 20% of all cancers worldwide in humans, this viral transformation represents a complex, multistage and multifactorial process. An example is the Epstein-Barr virus (EBV), a herpesvirus that latently infects over 90% of the population. Although the infection often courses asymptomatically, EBV is able to modify its genomic expression by establishing different latency phases, thus altering the B lymphocytes and epithelial cells metabolism, a determinant process in the appearance and development of different pathologies ranging from infectious mononucleosis to oncological processes such as Burkitt's lymphoma, gastric cancer and nasopharyngeal cancer.


Subject(s)
Humans , Herpesvirus 4, Human , Oncogenes , Viral Proteins , Gene Expression , Virus Latency
19.
Braz. j. microbiol ; Braz. j. microbiol;49(2): 336-346, Apr.-June 2018. tab, graf
Article in English | LILACS | ID: biblio-889223

ABSTRACT

Abstract Equine influenza is one of the major respiratory infectious diseases in horses. An equine influenza virus outbreak was identified in vaccinated and unvaccinated horses in a veterinary school hospital in São Paulo, SP, Brazil, in September 2015. The twelve equine influenza viruses isolated belonged to Florida Clade 1. The hemagglutinin and neuraminidase amino acid sequences were compared with the recent isolates from North and South America and the World Organisation for Animal Health recommended Florida Clade 1 vaccine strain. The hemagglutinin amino acid sequences had nine substitutions, compared with the vaccine strain. Two of them were in antigenic site A (A138S and G142R), one in antigenic site E (R62K) and another not in antigenic site (K304E). The four substitutions changed the hydrophobicity of hemagglutinin. Three distinct genetic variants were identified during the outbreak. Eleven variants were found in four quasispecies, which suggests the equine influenza virus evolved during the outbreak. The use of an out of date vaccine strain or updated vaccines without the production of protective antibody titers might be the major contributing factors on virus dissemination during this outbreak.


Subject(s)
Animals , Genetic Variation , Disease Outbreaks , Orthomyxoviridae Infections/veterinary , Evolution, Molecular , Influenza A Virus, H3N8 Subtype/isolation & purification , Horse Diseases/epidemiology , Horse Diseases/virology , Orthomyxoviridae , Viral Proteins/genetics , Brazil/epidemiology , Sequence Analysis, DNA , Orthomyxoviridae Infections/epidemiology , Orthomyxoviridae Infections/virology , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Amino Acid Substitution , Influenza A Virus, H3N8 Subtype/classification , Influenza A Virus, H3N8 Subtype/genetics , Genotype , Horses , Hospitals, Animal , Neuraminidase/genetics
20.
Arch. cardiol. Méx ; Arch. cardiol. Méx;88(5): 503-506, dic. 2018. graf
Article in Spanish | LILACS | ID: biblio-1142162

ABSTRACT

Resumen El desarrollo de hipertensión arterial pulmonar asociada al virus de inmunodeficiencia humana reduce la probabilidad de sobrevivencia en el paciente afectado en comparación con el que no presenta esta alteración cardiopulmonar. La fisiopatogenia aún es incierta. Existen varias líneas de investigación para asociar las diferentes proteínas del virus en la lesión endo- telial. Desde el punto de vista terapéutico, existen modalidades de tratamiento que permiten una expectativa de vida aceptable.


Abstract The development of pulmonary arterial hypertension associated with human immunodeficiency virus reduces the probability of survival in the patient affected compared to those without cardiopulmonary disease. The pathophysiology is uncertain. There are several lines of research to associate the different proteins of the virus in the endothelial lesion. From a therapeutic point of view there are treatment modalities that allow an acceptable life expectancy.


Subject(s)
Humans , Viral Proteins/metabolism , HIV Infections/complications , Hypertension, Pulmonary/etiology , HIV Infections/mortality , Life Expectancy , Hypertension, Pulmonary/physiopathology , Hypertension, Pulmonary/therapy
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