Current understanding of an Emerging Coronavirus usingin silico approach: Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) / Compreensão atual de um coronavírus emergente usando abordagem in silico: síndrome respiratória aguda grave - coronavírus-2 (SARS-CoV-2)

Novel coronavirus (nCoV) namely "SARS-CoV-2" is being found responsible for current PANDEMIC commenced from Wuhan (China) since December 2019 and has been described with epidemiological linkage to China in about 221 countries and territories until now. In this study we have characterized the genetic lineage of SARS-CoV-2 and report the recombination within the genus and subgenus of coronaviruses. Phylogenetic relationship of thirty nine coronaviruses belonging to its four genera and five subgenera was analyzed by using the Neighbor-joining method using MEGA 6.0. Phylogenetic trees of full length genome, various proteins (spike, envelope, membrane and nucleocapsid) nucleotide sequences were constructed separately. Putative recombination was probed via RDP4. Our analysis describes that the "SARS-CoV-2" although shows great similarity to Bat-SARS-CoVs sequences through whole genome (giving sequence similarity 89%), exhibits conflicting grouping with the Bat-SARS-like coronavirus sequences (MG772933 and MG772934). Furthermore, seven recombination events were observed in SARS-CoV-2 (NC_045512) by RDP4. But not a single recombination event fulfills the high level of certainty. Recombination mostly housed in spike protein genes than rest of the genome indicating breakpoint cluster arises beyond the 95% and 99% breakpoint density intervals. Genetic similarity levels observed among "SARS-CoV-2" and Bat-SARS-CoVs advocated that the latter did not exhibit the specific variant that cause outbreak in humans, proposing a suggestion that "SARS-CoV-2" has originated possibly from bats. These genomic features and their probable association with virus characteristics along with virulence in humans require further consideration.

O novo coronavírus (nCoV), nomeadamente "SARS-CoV-2", foi considerado responsável pela pandemia atual iniciada em Wuhan (China) desde dezembro de 2019 e foi descrito com ligação epidemiológica à China em cerca de 221 países e territórios até agora. Neste estudo, caracterizamos a linhagem genética do SARS-CoV-2 e relatamos a recombinação dentro do gênero e subgênero dos coronavírus. A relação filogenética de 39 coronavírus pertencentes a seus quatro gêneros e cinco subgêneros foi analisada usando o método de Neighbour-joining usando MEGA 6.0. Árvores filogenéticas do genoma de comprimento total, várias proteínas (espícula, envelope, membrana e nucleocapsídeo), sequências de nucleotídeos foram construídas separadamente. A recombinação putativa foi testada via RDP4. Nossa análise descreve que o "SARS-CoV-2", embora mostre grande semelhança com as sequências de Bat-SARS-CoVs em todo o genoma (dando semelhança de sequência de 89%), exibe agrupamento conflitante com as sequências de coronavírus do tipo Bat-SARS (MG772933 e MG772934) Além disso, sete eventos de recombinação foram observados em SARS-CoV-2 (NC045512) por RDP4. Mas nem um único evento de recombinação preenche o alto nível de certeza. A recombinação está alojada mais em genes de proteína de pico, principalmente, do que no resto do genoma, indicando que o cluster de ponto de interrupção surge além dos intervalos de densidade de ponto de interrupção de 95% e 99%. Os níveis de similaridade genética observados entre "SARS-CoV-2" e Bat-SARS-CoVs defendem que o último não exibe a variante específica que causa surto em humanos, sugerindo que "SARS-CoV-2" tenha se originado possivelmente de morcegos. Essas características genômicas e sua provável associação com as características do vírus, juntamente com a virulência em humanos, requerem uma consideração mais aprofundada.

Genes humanos asociados a la infección por el SARS-CoV-2 / Human genes associated with SARS-CoV-2 infection

RESUMEN Fundamento: La variabilidad clínica de la infección por el SARS-CoV-2 se debe, en parte, a factores genéticos. Objetivo: Describir los principales genes de susceptibilidad a la Covid-19. Metodología: Se realizó una revisión bibliográfica en Google Académico, SciELO, Annual Reviews y PubMed Central. Los descriptores que se utilizaron para la búsqueda de los documentos fueron consultados en el DeCS, estos fueron: SARS-CoV-2, Covid-19, genética y predisposición genética a la enfermedad. Se seleccionaron artículos disponibles a texto completo en inglés y en español, preferentemente de revistas arbitradas por pares. Resultados: Entre los genes implicados en la infección por el SARS-CoV-2 se encuentran DDX1 que promueve la replicación viral, IFITM1, IFITM2, IFITM3, IFNAR2 que codifican proteínas inducidas por el interferón, los genes de receptores (ACE2, ANPEP, DPP4), los genes de proteasas (TMPRSS2, furin, TMPRSS11D, CTSL, CTSB) que contribuyen a la entrada viral, genes de la respuesta inmune como ABO y metalopeptidasas como la familia ADAM. Se han detectado polimorfismos genéticos de riesgo. Conclusiones: En la infección por el SARS-CoV-2 se produce una compleja interrelación entre factores ambientales y genéticos que determinan la susceptibilidad de las personas a la Covid y su gravedad. El papel de los genes en la susceptibilidad a la Covid-19 deberá continuar investigándose.

ABSTRACT Background: The clinical variability of SARS-CoV-2 infection is partially due to genetic factors. Objective: To describe the main Covid-19 susceptibility genes. Methodology: A literature review was performed in Google Scholar, SciELO, Annual Reviews and PubMed Central. The descriptors used to search the documents were consulted in DeCS: SARS-CoV-2, Covid-19, genetics and genetic predisposition to disease. Full text articles available in English and Spanish were selected, rather from peer-reviewed journals. Results: Genes involved in SARS-CoV-2 infection include DDX1 which promotes viral replication, IFITM1, IFITM2, IFITM3, IFNAR2 encoding interferon-induced proteins, receptor genes (ACE2, ANPEP, DPP4), protease genes (TMPRSS2, furin, TMPRSS11D, CTSL, CTSB) that contribute to viral entry, immune response genes such as ABO and metallopeptidases such as the ADAM family. Risk genetic polymorphisms have been detected. Conclusions: In SARS-CoV-2 infection, there is a complex interaction between environmental and genetic factors that determine the susceptibility of individuals to Covid and its severity. The role of genes in Covid-19 susceptibility should be further investigated.

Vacunas para SARS-CoV-2: diferentes estrategias de los desarrollos en curso / Vaccines for SARS-CoV-2: different strategies of ongoing developments

El objetivo de este artículo es proporcionar una guía que sirva para la interpretación y seguimiento de los esfuerzos que se están desarrollando en todo el mundo con el objetivo de obtener una vacuna que pueda generar inmunidad contra el nuevo coronavirus SARS-CoV-2 de 2019, el agente causante de la enfermedad por coronavirus denominada COVID-19. Cinco meses después de haber sido detectada la enfermedad, ya hay 102 vacunas en distintos estadios de desarrollo, registradas por la Organización Mundial de la Salud (OMS), correspondientes a 8 plataformas vacunales con diferentes estrategias, y todos los días aparecen nuevas. Esto representará un enorme desafío de organismos internacionales, para la evaluación, comparación y selección de aquellas que cumplan con los criterios regulatorios indispensables de seguridad y eficacia y que, por otro lado, puedan ser producidas en cantidades suficientes para abastecer la demanda mundial. (AU)

The objective of this article is to provide a guide to help the interpretation and monitoring the efforts that are being carried out worldwide to obtain a vaccine that will be able to generate immunity against the new 2019 SARS-CoV-2 coronavirus, the viral agent causes the disease named COVID-19. Five months after the disease was detected, there are already 102 vaccines at different stages of development, registered by World Health Organization (WHO), corresponding to 8 vaccination platforms base on different strategies, and every day new ones appear. This will represent a huge challenge for international organizations, to evaluate, compare and selects those that will meet the essential regulatory criteria of safety and efficacy and that, would be able to be produced in enough quantities to supply the worldwide demand. Key words: SARS-Cov-2 vaccine, vaccine platform, COVID-19 strategy, attenuated virus, viral vector, viral proteins, viral DNA, viral RNA, nucleic acids, viral like particles, WHO. (AU)

Production of specific antibodies against SARS-coronavirus nucleocapsid protein without cross reactivity with human coronaviruses 229E and OC43

Severe acute respiratory syndrome (SARS) is a life-threatening disease for which accurate diagnosis is essential. Although many tools have been developed for the diagnosis of SARS, false-positive reactions in negative sera may occur because of cross-reactivity with other coronaviruses. We have raised polyclonal and monoclonal antibodies (Abs) using a recombinant form of the SARS virus nucleocapsid protein. Cross-reactivity of these anti-SARS Abs against human coronavirus (HCoV) 229E and HCoV OC43 were determined by Western blotting. The Abs produced reacted with recombinant SARS virus nucleocapsid protein, but not with HCoV 229E or HCoV OC43.

Comparative study of synonymous codon usage variations between the nucleocapsid and spike genes of coronavirus, and C-type lectin domain genes of human and mouse

Coronaviruses (CoVs) are single-stranded RNA viruses which contain the largest RNA genomes, and severe acute respiratory syndrome coronavirus (SARS-CoV), a newly found group 2 CoV, emerged as infectious disease with high mortality rate. In this study, we compared the synonymous codon usage patterns between the nucleocapsid and spike genes of CoVs, and C-type lectin domain (CTLD) genes of human and mouse on the codon basis. Findings indicate that the nucleocapsid genes of CoVs were affected from the synonymous codon usage bias than spike genes, and the CTLDs of human and mouse partially overlapped with the nucleocapsid genes of CoVs. In addition, we observed that CTLDs which showed the similar relative synonymous codon usage (RSCU) patterns with CoVs were commonly derived from the human chromosome 12, and mouse chromosome 6 and 12, suggesting that there might be a specific genomic region or chromosomes which show a more similar synonymous codon usage pattern with viral genes. Our findings contribute to developing the codon-optimization method in DNA vaccines, and further study is needed to determine a specific correlation between the codon usage patterns and the chromosomal locations in higher organisms.

Expression and purification of SARS coronavirus membrane protein / 华中科技大学学报（医学）（英德文版）

To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.