Emergence of novel SARS-CoV-2 lineages are under the spotlight of the media, scientific community and governments. Recent reports of novel variants in the United Kingdom, South Africa and Brazil (B.1.1.28-E484 K) have raised intense interest because of a possible higher transmission rate or resistance to the novel vaccines. Nevertheless, the spread of B.1.1.28 (E484 K) and other variants in Brazil is still unknown. In this work, we investigated the population structure and genomic complexity of SARS-CoV-2 in Rio Grande do Sul, the southernmost state in Brazil. Most samples sequenced belonged to the B.1.1.28 (E484 K) lineage, demonstrating its widespread dispersion. We were the first to identify two independent events of co-infection caused by the occurrence of B.1.1.28 (E484 K) with either B.1.1.248 or B.1.91 lineages. Also, clustering analysis revealed the occurrence of a novel cluster of samples circulating in the state (named VUI-NP13 L) characterized by 12 lineage-defining mutations. In light of the evidence for E484 K dispersion, co-infection and emergence of VUI-NP13 L in Rio Grande do Sul, we reaffirm the importance of establishing strict and effective social distancing measures to counter the spread of potentially more hazardous SARS-CoV-2 strains.

COVID-19 (Coronavirus Disease 2019) outbreak was declared a pandemic, byWorld Health Organization, on March 11, 2020. Viral detection using RT-qPCR has been among the most important factors helping to control local spread of SARS-CoV-2 and it is considered the "gold standard" for diagnosis. Nevertheless, the RNA extraction step is both laborious and expensive, thus hampering the diagnosis in many places where there are not laboratory staff of funds enough to contribute for diagnosis efforts.Thus, the need to simplify procedures, reduce costs of the techniques used, and expand the capacity of the number of diagnostics of COVID-19 is imperative. In this study, detection of SARS-CoV-2 in the absence of RNA extraction has been successfully achieved through pre-treatment of the clinical sample with Proteinase K.The results show that only the use of proteinase K, without the need to perform the whole standard protocol for sample extraction and purification, can be an efficient technique for the diagnosis of COVID-19, since 91% of the samples matched the results with the standard procedure, with an average increase of 5.64 CT in the RT-qPCR.

The World Health Organization (WHO) has declared a pandemic of COVID-19, the disease caused by the recently described SARS-CoV-2. The relevance and importance of mass diagnosis in order to find the asymptomatic individuals is widely recognized as a mandatory tool to reinforce the control measures for monitoring virus circulation and reduce the spreading of SARS-CoV-2. Here, we described quickness and cheaper strategies of direct RT-qPCR (in the absence of RNA isolation) and compared the results to those obtained using standard RNA isolation procedure. The tests varied using pure, diluted samples, combined with Proteinase K (PK) or Lysis Buffer. Our findings showed consistently that PK pre-treated samples in the absence of RNA extraction procedures presents similar results to those obtained by standard RNA isolation procedures. On average, 16 samples extracted with the MagMAX™ CORE Kit, take around 2 hours, costing an average of USD 5, the pre-treatment of samples using PK, on the other hand, would cut the value to less than USD 0.30 and reduce the time of procedure in more than 1 ½ hours. The present study suggests the use of PK treatment instead of RNA isolation in order to reduce costs and time in processing samples for molecular diagnosis of SARS-CoV-2.

In this study, serial dilutions of SARS-CoV 2 RNA extract were tested using RT-dPCR using three different primer-probe assays aiming SARS-CoV 2 nucleocapsid coding region. Narrower confidence intervals, indicating high quantification precision were obtained in 100 and 1000-fold serial dilution and RT-dPCR results were equivalent between different assays in the same dilution. High accuracy of this test allowed conclusions regarding the ability of this technique to evaluate precisely the amount of genomic copies present in a sample. We believe that this fast and safe method can assist other researchers in titration of SARS-CoV2 controls used in RT-qPCR without the need of virus isolation.

Emergence of novel SARS-CoV-2 lineages are under the spotlight of the media, scientific community and governments. Recent reports of novel variants in the United Kingdom, South Africa and Brazil (B.1.1.28-E484K) have raised intense interest because of a possible higher transmission rate or resistance to the novel vaccines. Nevertheless, the spread of B.1.1.28 (E484K) and other variants in Brazil is still unknown. In this work, we investigated the population structure and genomic complexity of SARS-CoV-2 in Rio Grande do Sul, the southernmost state in Brazil. Most samples sequenced belonged to the B.1.1.28 (E484K) lineage, demonstrating its widespread dispersion. We were the first to identify two independent events of co-infection caused by the occurrence of B.1.1.28 (E484K) with either B.1.1.248 or B.1.91 lineages. Also, clustering analysis revealed the occurrence of a novel cluster of samples circulating in the state (named VUI-NP13L) characterized by 12 lineage-defining mutations. In light of the evidence for E484K dispersion, co-infection and emergence of VUI-NP13L in Rio Grande do Sul, we reaffirm the importance of establishing strict and effective social distancing measures to counter the spread of potentially more hazardous SARS-CoV-2 strains.HighlightsThe novel variant B.1.1.28 (E484K) previously described in Rio de Janeiro is currently spread across the southernmost state of Brazil;The novel variant VUI-NP13L was also identified by causing a local outbreak in Rio Grande do Sul;B.1.1.28 (E484K) is able to establish successful coinfection events co-occurring simultaneously with different lineages of SARS-CoV-2.

December 2019 marked the begining of the greatest pandemic since Spanish Flu, the disease named Covid-19 that cause severe pneumonia. Until May 19, 2020 more than 4 million and 700 thousand cases were oficially notified with about 316 thousand deaths.Etiological agent of the disease was identified as being a new coronavirus, Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2). In this study we compared four different manual methods for RNA isolation and purification for detection of SARS-CoV-2 through qRT-PCR, as well as the extraction quality itself through detection of RNAse P. Magnetic beads-based (MagMax ™ ) and silica column-based (Biopur ® ) methods presented the better performances. Concerning to the mean delay in CT values when compared to MagMax ™ , TRIzol ™ , Biopur ® and EasyExtract presented 0,39, 0,95 and 5,23 respectively. Agreement between positive and negative results of different methods when compared with the one with better performance MagMax™ was 94,44% for silica column-based method (Biopur®), 88,89% for phenol-chroloform-based method (TRIzol™) and 77,78% for EasyExtract. We aimed to evaluate how reliable each method is for diagnostic purposes and to propose alternatives when usual methods are not available. In this regard, magnectic beads and silica column-based methods are convenient and reliable choices and phenol-chloroform-based method could also be chosen as an alternative.

Yellow Fever Virus (YFV) and Chikungunya Virus (CHIV) are neglected reemerging pathogens that cause comorbidities worldwide. Since no antiviral drug is prescribed for those infections, there is a demand on researching compounds that inhibit viral replication. Saponins are amphiphilic compounds that already demonstrated in vitro activity against enveloped virus. Therefore, two purified saponin fractions from Quillaja spp. were evaluated regarding their antiviral potential against YFV and CHIKV. The cell line used in this study was VERO (African green monkey kidney cells) since it is permissive to the replication of both viruses. The antiviral activity of both saponins fractions was screened using the plaque reduction assay protocol. Although saponins did not inhibited YFV replication, they strongly inhibited CHIKV. To confirm the absence of antiviral activity of Quillaja saponins against YFV, the cytopathic effect inhibition assay was performed also. Further studies are required to determine the antiviral mechanisms involved in the CHIKV inhibition.

Different approaches are in use to improve our knowledge about the causative agent of coronavirus disease (COVID-19). Cell culture-based methods are the better way to perform viral isolation, evaluate viral infectivity, and amplify the virus. Furthermore, next-generation sequencing (NGS) have been essential to analyze a complete genome and to describe new viral species and lineages that have arisen over time. Four naso-oropharyngeal swab samples, collected from April to July of 2020, were isolated and sequenced aiming to produce viral stocks and analyze the mutational profile of the found lineage. B.1.1.33 was the lineage detected in all sequences. Although the samples belong to the same lineage, it was possible to evaluate different mutations found including some that were first described in these sequences, like the S:H655Y and T63N. The results described here can help to elicit how the pandemic started to spread and how it has been evolving in south Brazil.