Aspectos generales sobre la estructura del coronavirus del síndrome respiratorio agudo grave 2 (SARS-CoV-2) / General aspects about the structure of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Introduction: In late 2019, a new coronavirus named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that causes respiratory-related illness was reported in Wuhan, China. This virus can attack human lung cells causing a disease called coronavirus disease 2019 (COVID-19), which can lead to pneumonia and acute respiratory distress syndrome. Objective: Describe the structural characteristics of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Methods: A review was written from 47 bibliographic references. Articles and information from national and international journals available in the PubMed, Scopus, Medline, SciELO databases were used. The quality, reliability and validity of the selected articles were analyzed to carry out an adequate review. Analysis-synthesis and logical deduction methods were applied. Development: An introduction to the general aspects of the structure of SARS-CoV-2 is provided by stating the characteristics of the structural and non-structural proteins encoded by the viral genome, which provides the basis for understanding viral entry mechanisms to the host cell, and may be useful to stimulate the search for novel insights and possible therapeutic targets to fight the infection. Conclusions: Knowledge of the structure of the SARS-CoV-2 virus and the characteristics of the structural and non-structural proteins provides the basis for understanding the viral mechanisms of infection and the strategies for developing effective therapeutics(AU)

Introducción: A finales de 2019 se informó el brote de un nuevo coronavirus en Wuhan, China, llamado Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) que causa alteraciones en el aparato respiratorio. Este virus puede atacar las células humanas del pulmón causando una enfermedad denominada enfermedad por coronavirus 2019 (COVID-19), que puede producir neumonía y un síndrome de dificultad respiratoria aguda. Objetivo: Describir las características estructurales del virus SARS-CoV-2. Métodos: Se realizó una revisión bibliográfica a partir de 47 referencias. Se utilizaron artículos e información de revistas nacionales e internacionales disponibles en las bases de datos PubMed, Scopus, Medline, SciELO. Para llevar a cabo una revisión adecuada, se analizaron la calidad, fiabilidad y validez de los artículos seleccionados. Se aplicaron métodos de análisis-síntesis y deducción lógica. Desarrollo: Se proporciona una introducción de los aspectos generales de la estructura del SARS-CoV-2. Se enuncian las características de las proteínas estructurales y no estructurales codificadas por el genoma viral, lo que provee la base para comprender los mecanismos virales de entrada a la célula huésped. El artículo resulta de utilidad para estimular la búsqueda de nuevos conocimientos y posibles objetivos terapéuticos para combatir la infección. Conclusiones: El conocimiento sobre la estructura del virus SARS-CoV-2 y las características de las proteínas estructurales y no estructurales que lo forman ampara significativamente las bases para entender los mecanismos virales de la infección y las estrategias para el desarrollo terapéutico efectivo(AU)

Research progress and application of nanopore sequencing technology / 生物工程学报

Sequencing technology has been greatly improved in terms of throughput and cost. The single-molecule nanopore DNA sequencing, one of the major branches of the third-generation sequencing technology, has made great contributions in the fields of medicine and life sciences due to its advantages of ultra-long reading length, real-time detection and direct detection of base methylation modification, etc. This article briefly describes the principle of nanopore sequencing technology, and discusses its application in clinical, animal, plant, bacterial and virus fields and its future development direction.

Characterisation of a rare, reassortant human G10P[14] rotavirus strain detected in Honduras

BACKGROUND Although first detected in animals, the rare rotavirus strain G10P[14] has been sporadically detected in humans in Slovenia, Thailand, United Kingdom and Australia among other countries. Earlier studies suggest that the strains found in humans resulted from interspecies transmission and reassortment between human and bovine rotavirus strains. OBJECTIVES In this study, a G10P[14] rotavirus genotype detected in a human stool sample in Honduras during the 2010-2011 rotavirus season, from an unvaccinated 30-month old boy who reported at the hospital with severe diarrhea and vomiting, was characterised to determine the possible evolutionary origin of the rare strain. METHODS For the sample detected as G10P[14], 10% suspension was prepared and used for RNA extraction and sequence independent amplification. The amplicons were sequenced by next-generation sequencing using the Illumina MiSeq 150 paired end method. The sequence reads were analysed using CLC Genomics Workbench 6.0 and phylogenetic trees were constructed using PhyML version 3.0. FINDINGS The next generation sequencing and phylogenetic analyses of the 11-segmented genome of the G10P[14] strain allowed classification as G10-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3. Six of the genes (VP1, VP2, VP3, VP6, NSP2 and NSP4) were DS-1-like. NSP1 and NSP5 were AU-1-like and NSP3 was T6, which suggests that multiple reassortment events occurred in the evolution of the strain. The phylogenetic analyses and genetic distance calculations showed that the VP7, VP4, VP6, VP1, VP3, NSP1, NSP3 and NSP4 genes clustered predominantly with bovine strains. NSP2 and VP2 genes were most closely related to simian and human strains, respectively, and NSP5 was most closely related to a rhesus strain. MAIN CONCLUSIONS The genetic characterisation of the G10P[14] strain from Honduras suggests that its genome resulted from multiple reassortment events which were possibly mediated through interspecies transmissions.

Application of nanopore sequencing technology in viral genome research / 中华实验和临床病毒学杂志

MinION is a commercial nanopore sequencer developed by Oxford Nanopore Techno-logies. Compared with the second-generation sequencing platform, MinION is characterized by ultra-long reads, simple and fast sequencing, high portability and real-time data analysis. MinION has been widely applied to the fields of virus identification, whole genome sequencing, new virus discovery, quasispecies and virus evolution. In this article, we review the application of nanopore sequencing technology in the research of the viral genome in recent years, and discuss the existing problems. We also present the prospect for the development trend of nanopore sequencing in the future.

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes.</p><p><b>METHODS</b>In this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing.</p><p><b>RESULTS</b>Within the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run.</p><p><b>CONCLUSION</b>MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.</p>

Virus chikungunya: Características virales y evolución genética / Chikungunya Virus: Viral Features and Genetic Evolution

Resumen El virus chikungunya pertenece al género Alphavirus, de la familia de los Togaviridae. Es transmitido por artrópodos, en particular por la picada de especies de mosquitos, tales como Aedes aegypti y Aedes albopictus. El curso clínico característico de la infección incluye fiebres, artralgias y exantema. Desde que fue reportado en 1952 en los límites de Tanzania y Mozambique ha generado brotes de enorme significado epidemiológico. Recientemente fue causado un brote en las Américas por una cepa del virus, aparentemente, asiática. En esta revisión presentamos su filogenia, estructura y organización del genoma. Enfatizaremos en el mecanismo de multiplicación y la expresión genética. Finalmente, la interacción virus-huésped y sus mecanismos de adaptación a vectores específicos también son discutidos.

Abstract Chikungunya virus belongs to the Alphavirus genus of the family Togaviridae. It is transmitted by arthropods, in particular by the biting of mosquito species such as Aedes aegypti and Aedes albopictus. The characteristic clinical course of the infection includes fever, arthralgia, and rash. Since it was reported on 1952 on the borders of Tanzania and Mozambique, it has been triggered outbreaks with tremendous epidemiological significance. Recently an outbreak was caused in the Americas by an apparent Asian strain of this virus. In this review we present its phylogeny, structure and genome organization. We will emphasize the mechanism of replication and gene expression. Finally, the virus-host interaction and its mechanisms of adaptation to specific vectors are also discussed.

Analysis on relation between viral genome with hepatitis B virus e antigen and liver function in chronic hepatitis B patients / 国际检验医学杂志

Objective To study the relationship among hepatitis B viral genome (HBV‐DNA) ,hepatitis Be antigen(HBeAg) and liver function in chronic hepatitis B patients ,and to provide the reference for clinical treatment .Methods The quantitative levels of HBV‐DNA ,HBeAg ,alanine aminotransferase (ALT) and aspartate aminotransferase(AST) in 401 patients were analyzed and the correlation analysis between HBV‐DNA and HBeAg was performed .The grouping was performed according to the HBV‐DNA and HBeAg quantitative levels and the differences of ALT and AST levels were compared among the groups .Results (1) The correla‐tion existed between HBV‐DNA and HBeAg positive rate ,r=0 .671(P<0 .01);(2)when HBV‐DNA load reaching 105 copies/mL , serum ALT and AST levels showed significantly increased compared with the HBV‐DNA negative group and low load group ,the difference was statistically significant (P<0 .05);(3)when HBV‐DNA load was equivalent ,the difference of ALT and AST activity had no statistically significant difference between the HBeAg‐positive and HBeAg‐negative groups .Conclusion (1)HBeAg has a correlation with HBV‐DNA ;(2)the patients with higher HBV‐DNA load are easy to develop the liver function abnormality ;(3)the HbeAg existence situation has no obvious relation with the liver function .

Zika virus: Genome sequence analysis and nucleic acid detection methods / 第二军医大学学报

Objective To analyze the genome sequence characteristics of Zika virus and to develop nucleic acid detection methods for Zika virus. Methods The phylogenetic tree of 81 kinds of Flavivirus was constructed. The differences of nucleotide and amino acid sequence among Zika virus, type 4 dengue and Japanese encephalitis virus (JEV) were analyzed and compared. The gene mutated sites of Asian and African Zika virus, especially four Zika virus strains from China, were analyzed. A set of primers and probes of real-time quantitative PCR for Zika virus were designed after comparing the genome sequences of Asian and African Zika virus. Results Spondweni and Kedougou viruses were the closest homologously to Zika virus among 81 kinds of Flavivirus. Comparison of full genomic nucleic acid sequence showed that Zika virus was closer to type 4 dengue virus than JEV, whereas comparison of amino acid yielded an opposite result. Compared with traditional Asian type Zika virus, Guangdong GD01 strains had 5 amino acid mutated sites, Zhejiang ZJ03 strains had 6 mutated sites, and VE Ganxian strains had 33 mutated sites. Detection of designed PCR primers and probes for plasmid RNA was positive, with the lower limit of detection being 100 copies/mL and Zika virus RNA was detected to be positive, type 1-4 dengue virus and Japanese encephalitis virus being negative. Conclusion Zika virus and Spondweni virus are the closest homologously. The high mutation character of VE Ganxian strains indicates that Zika might evolve fast. PCR primers and probes designed in this paper can be used for Asian and African type Zika virus detection, with relatively higher sensitivity and specificity.

Improved assembly procedure of viral RNA genomes amplified with Phi29 polymerase from new generation sequencing data

BACKGROUND: New sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes. FINDINGS: Three different assembly softwares (ABySS, Ray and SPAdes) were tested for their ability to correctly assemble the full-length of viral genomes. Although in all cases, our pre-processed method improved genome assembly, only its combination with the use of SPAdes allowed us to obtain the full-length of the viral genomes tested in one contig. CONCLUSIONS: The proposed pipeline is able to overcome drawbacks due to the generation of chimeric reads during the amplification of viral RNA which considerably improves the assembling of full-length viral genomes.

Characteristics of genome variations of ebola viruses in 2014 epidemic / 第二军医大学学报

Objective To explore the relationship between the Ebola virus genome variations and its epidemiological characteristics by analyzing the 102 whole genome sequences of Ebola viruses in 2014 outbreak. Methods Whole genome sequences of Ebola viruses (EBOVs) were obtained from the NCBI database, and the variations in genome sequences were analyzed by Mummer3. 0. The evolutionary analysis was carried out through MEGA5; and the 3D modeling of the protein was performed using CPHmodels and PyMOL software. Results It was found that there were 606 single nucleotide variants (SNVs) in the genome of 2014 EBOVs, of which 49 nonsynonymous SNVs were unique. The amino acids of NP-182, GP-82 and L-1951, which were highly conserved not only among all the Zaire EBOVs before 2014, but also among different EBOV species, were altered in 2014 EBOVs. Conclusion The unique mutation of 2014 EBOVs resulting in alterations of NP, GP and L protein, especially the alteration of aa82 of GP (Ala→Val), might weaken the stability of α-helix where the amino acid is located, which might be associated with the weakened lethality and enhanced transmission of the virus. Further studies are needed to confirm whether genomic variations in 2014 EBOVs is responsible for change of the epidemiological characteristics.

Molecular modeling and conformational analysis of native and refolded viral genome-linked protein of Cardamom mosaic virus.

The viral genome-linked protein (VPg) of Potyviruses is covalently attached to the 5’ end of the genomic RNA. Towards biophysical characterization, the VPg coding region of Cardamom mosaic virus (CdMV) was amplified from the cDNA and expressed in E. coli. Most of the expressed VPg aggregated as inclusion bodies that were solubilized with urea and refolded with L-arginine hydrochloride. The various forms of CdMV VPg (native, denatured and refolded) were purified and the conformational variations between these forms were observed with fluorescence spectroscopy. Native and refolded CdMV VPg showed unordered secondary structure in the circular dichroism (CD) spectrum. The model of CdMV VPg was built based on the crystal structure of phosphotriesterase (from Pseudomonas diminuta), which had the maximum sequence homology with VPg to identify the arrangement of conserved amino acids in the protein to study the functional diversity of VPg. This is the first report on the VPg of CdMV, which is classified as a new member of the Macluravirus genus of the Potyviridae family.

Recombination analysis of full-length genomic sequences of novel influenza virus A/H1N1 in 2009 pandemic / 第二军医大学学报

Objective: To analyze the recombination of full-length genomic sequences of novel influenza virus A/H1N1 in 2009 pandemic. Methods: The full-length sequences of the novel A/H1N1 and reference sequences were downloaded from NCBI database. MEGA4.0 software was used to connect, align sequences, and analyze the similarity between the full-length sequences of the novel virus and each of the reference strains. Recombination was analyzed by Simplot software (version 3.5.1). Results: Simplot analysis indicated that the PB1 genes (polymerase B1, PB1) of the novel A/H1N1 viruses might evolve from human H3N2 virus (identity: 93.7%); the PB2 genes (polymerase B2, PB2) and the PA genes (polymerase A, PA) might evolve from avian H5N1 viruses (identity: 89.0%, 89.9%, respectively); the HA genes (hemagglutinin, HA), the NP genes (nucleoprotein, NP) and the NS genes (non-structural protein, NS) showed high similarities with those of swine H1N1 viruses isolated in North America (identity: 91.7%, 93.1%, and 93.1%, respectively); and the NA genes (neuraminidase, NA) and the MP genes (matrix protein, MP) might evolve from European swine H1N1 viruses (identity: 90.5%, 95.5%, respectively). The full-length sequence of the novel A/H1N1 viruses had a highest similarities with swine H1N1 viruses isolated in North America (identity: 83.9%). Conclusion: The novel influenza virus A/H1N1 is a recombinant virus evolving from human H3N2 viruses, swine H1N1 from North America, swine H1N1 from Europe, and swine H5N1 from Asia.

Alta prevalência do genótipo 1 em portadores de hepatite C crônica em Belo Horizonte, MG / High prevalence of genotype 1 in individuals with hepatitis C in Belo Horizonte, MG

O vírus da hepatite C é caracterizado pela significativa heterogeneidade genética e é atualmente classificado em seis genótipos principais e diversos subtipos. A determinação do genótipo do vírus tem importância na prática clínica para orientar o tratamento dos pacientes portadores de hepatite C crônica. A prevalência dos diferentes genótipos e subtipos do vírus da hepatite C não tem sido amplamente estudada em algumas regiões do Brasil. Neste estudo foram analisadas 788 amostras de pacientes portadores de hepatite C crônica atendidos nos Centros de Referência em Hepatites Virais de Belo Horizonte, entre 2002 e 2006. A genotipagem do vírus foi realizada por seqüenciamento direto da região 5’ UTR. Adicionalmente, foi realizada análise filogenética incluindo todas as variantes genotípicas obtidas. Observou-se alta prevalência do genótipo 1 (78,4 por cento; 1b [40,4 por cento], 1a [37,5 por cento] e 1a/b [0,5 por cento]), seguida pelo genótipo 3a (17,9 por cento) e pelo 2b (3,1 por cento). Foram identificadas três amostras (0,4 por cento) com o genótipo 2a/c e duas amostras (0,2 por cento) com o genótipo 4. A análise filogenética mostrou a segregação esperada das seqüências obtidas junto às seqüências de referência para os genótipos 1, 2, 3 e 4, exceto em duas amostras do genótipo 1a. A alta prevalência do genótipo 1 (78,4 por cento), encontrada na população de Belo Horizonte é semelhante à previamente descrita em outras cidades, como Rio de Janeiro, mas superior à encontrada em São Paulo e no Sul do país. A presença de raras seqüências atípicas da região 5’UTR sugere a presença de variantes do vírus da hepatite C nesta população.

The hepatitis C virus is characterized by significant genetic heterogeneity. It is currently classified into six main genotypes and several subtypes. Determining the genotype of the virus is important in clinical practice for guiding the treatment for individuals with chronic hepatitis C. The prevalence of different genotypes and subtypes of the hepatitis C virus has not been fully studied in some regions of Brazil. In this study, 788 samples from patients with chronic hepatitis C who were attended at the Viral Hepatitis Reference Centers in Belo Horizonte were analyzed between 2002 and 2006. The genotyping of the virus was performed by direct sequencing of the 5’ UTR region. Additionally, phylogenetic analysis was performed, including all of the genotypic variants obtained. High prevalence of genotype 1 (78.4 percent; 1b [40.4 percent], 1a [37.5 percent] and 1a/b [0.5 percent]) was observed, followed by genotypes 3a (17.9 percent) and 2b (3.1 percent). Three samples were identified as genotype 2a/c (0.4 percent) and two as genotype 4 (0.2 percent). The phylogenetic analysis showed the expected segregation of the sequences obtained, with regard to the reference sequences for genotypes 1, 2, 3 and 4, except for two samples of genotype 1a. The high prevalence of genotype 1 (78.4 percent) found in this population from Belo Horizonte was similar to previous reports from other cities such as Rio de Janeiro, but it was higher than what has been described in São Paulo and in the south of the country. The presence of rare atypical sequences from the 5’ UTR region suggests that variants in the hepatitis C virus exist in this population.

GTVseq: A Web-based Genotyping Tool for Viral Sequences

Genotyping Tool for Viral SEQuences (GTVseq) provides scientists with the genotype information on the viral genome sequences including HIV-1, HIV-2, HBV, HCV, HTLV-1, HTLV-2, poliovirus, enterovirus, flavivirus, Hantavirus, and rotavirus. GTVseq produces alternative and additive genotype information for the query viral sequences based on two different, but related, scoring methods. The genotype information produced is reported in a graphical manner for the reference genotype matches and each graphical output is linked to the detailed sequence alignments between the query and the matched reference sequences. GTVseq also reports the potential 'repeats' and/or 'recombination' sequence region in a separated window. GTVseq does not replace completely other well-known genotyping tools such as NCBI's virus sequence genotyping tool (http://www.ncbi. nlm.nih.gov/projects/genotyping/formpage.cgi), but provides additional information useful in the confirmation or for further investigation of the genotype(s) for the newly isolated viral sequences.

GTVseq: A Web-based Genotyping Tool for Viral Sequences

Genotyping Tool for Viral SEQuences (GTVseq) provides scientists with the genotype information on the viral genome sequences including HIV-1, HIV-2, HBV, HCV, HTLV-1, HTLV-2, poliovirus, enterovirus, flavivirus, Hantavirus, and rotavirus. GTVseq produces alternative and additive genotype information for the query viral sequences based on two different, but related, scoring methods. The genotype information produced is reported in a graphical manner for the reference genotype matches and each graphical output is linked to the detailed sequence alignments between the query and the matched reference sequences. GTVseq also reports the potential 'repeats' and/or 'recombination' sequence region in a separated window. GTVseq does not replace completely other well-known genotyping tools such as NCBI's virus sequence genotyping tool (http://www.ncbi. nlm.nih.gov/projects/genotyping/formpage.cgi), but provides additional information useful in the confirmation or for further investigation of the genotype(s) for the newly isolated viral sequences.

Detecção de roteiros e adenovírus na Grande São Paulo no período 1984-1986: estudo eletroforético do genoma dos rotavírus / Rotavirus and adenovirus detection in the Greater Säo Paulo in 1984-1986: eletrophoretic study of the rotavirus genome

No período de 1984 a 1986, 285 amostras de fezes de crianças com sintomatologia diarreica foram submetidas às provas diagnósticas de ensaio imunoenzimático, eletroforese em gel de poliacrilamida e microscopia eletrônica. Destas amostras, 15,4% foram positivas para rotavírus e 3,2% para adenovírus. Das 44 (15,4%) amostras positivas para rotavírus pelo método imunoenzimático, 37 apresentaram perfil eletroforético do RNA característico dos rotavírus. Destas últimas, 27 foram analisadas segundo o esquema de Lourenço et alii, 1981, tendo sido verificada grande heterogeneidade de perfis e predominância dos rota vírus do subgrupo 1 foi detectada (AU).