As part of any plan to lift or ease the confinement restrictions that are in place in many different countries, there is an urgent need to increase the capacity of laboratory testing for SARS CoV-2. Detection of the viral genome through RT-qPCR is the golden standard for this test, however, the high demand of the materials and reagents needed to sample individuals, purify the viral RNA, and perform the RT-qPCR test has resulted in a worldwide shortage of several of these supplies. Here, we show that directly lysed saliva samples can serve as a suitable source for viral RNA detection that is cheaper and can be as efficient as the classical protocol that involves column purification of the viral RNA. In addition, it surpasses the need for swab sampling, decreases the risk of the healthcare personnel involved in this process, and accelerates the diagnostic procedure.

The emergence and rapid increase of the B.1.1.7 (Alpha) lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) throughout the world were due to its increased transmissibility. However, it did not displace cocirculating lineages in most of Mexico, particularly B.1.1.519, which dominated the country from February to May 2021.

Alphaviruses have been the cause of both localized outbreaks and large epidemics of severe disease. Currently, there are no strategies or vaccines which are either safe or effective for preventing alphaviral infection or treating alphaviral disease. This deficit of viable therapeutics highlights the need to better understand the mechanisms behind alphaviral infection in order to develop novel antiviral strategies for treatment of alphaviral disease. In particular, this report details a previously uncharacterized aspect of the alphaviral life cycle: the importance of noncapped genomic viral RNAs for alphaviral infection. This offers new insights into the mechanisms of alphaviral replication and the impact of the noncapped genomic RNAs on viral packaging.

In many countries a second wave of infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has occurred, triggering a shortage of reagents needed for diagnosis and compromising the capacity of laboratory testing. There is an urgent need to develop methods to accelerate the diagnostic procedures. Pooling samples represents a strategy to overcome the shortage of reagents, since several samples can be tested using one reaction, significantly increasing the number and speed with which tests can be carried out. We have reported the feasibility to use a direct lysis procedure of saliva as source for RNA to SARS-CoV-2 genome detection by reverse transcription quantitative-PCR (RT-qPCR). Here, we show that the direct lysis of saliva pools, of either five or ten samples, does not compromise the detection of viral RNA. In addition, it is a sensitive, fast, and inexpensive method that can be used for massive screening, especially considering the proximity of the reincorporation of activities in universities, offices, and schools.

25Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe 26 disease and are a significant burden to public health. Alphaviral replication results in the 27 production of both capped and noncapped viral genomic RNAs, which are packaged 28 into virions during the infections of vertebrate and invertebrate cells. However, the roles 29 that the noncapped genomic RNAs (ncgRNAs) play during alphaviral infection have yet 30 to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral 31 infection was assessed by using mutants of the nsP1 protein of Sindbis virus (SINV), 32 which altered the synthesis of the ncgRNAs during infection by modulating the protein's 33 capping efficiency. Specifically, point mutants at residues Y286A and N376A decreased 34 capping efficiency, while a point mutant at D355A increased the capping efficiency of 35 the SINV genomic RNA during genuine viral infection. Viral growth kinetics were 36 significantly reduced for the D355A mutant relative to wild type infection, whereas the 37 Y286A and N376A mutants showed modest decreases in growth kinetics. Overall 38 genomic translation and nonstructural protein accumulation was found to correlate with 39 increases and decreases in capping efficiency. However, genomic, minus strand, and 40 subgenomic viral RNA synthesis was largely unaffected by the modulation of alphaviral 41 capping activity. In addition, translation of the subgenomic vRNA was found to be 42 unimpacted by changes in capping efficiency. The mechanism by which decreased 43 presence of ncgRNAs reduced viral growth kinetics was through the impaired 44 production of viral particles. Collectively, these data illustrate the importance of 45 ncgRNAs to viral infection and suggests that they play in integral role in the production 46 of viral progeny. 47 94 5' termini of the alphaviral genomic and subgenomic vRNAs, which provided the first 95 evidence for independent promoter initiation for the two positive-sense vRNA species 96 (9). While these studies were able to identify the presence of the 5' type-0 cap structure, 97 they were, by the nature of their design and technological limitations, unable to 98 determine the relative frequency with which the positive sense vRNAs were capped. 99Recently, we reported findings that indicated that the alphaviral genomic vRNAs 100 are not ubiquitously capped, and that a significant proportion of the genomic vRNAs 101 produced during SINV and RRV infection lack the 5' type-0 cap structure (18). 102 Furthermore, analyses of infectious and noninfectious viral particles demonstrated that 103 both the capped and noncapped genomic vRNAs are packaged into viral particles 104 throughout the course of infection. Through the use of tissue culture models of 105 alphaviral infection, the presence of the noncapped vRNAs were found to correlate with 106 the activation of a type-I IFN response. Moreover, an attenuated RRV mutant was found 107to produce fewer noncapped genomic RNAs relative to wild type virulent RRV (18...

AbstractAs part of any plan to lift or ease the confinement restrictions that are in place in many different countries, there is an urgent need to increase the capacity of laboratory testing for SARS CoV-2. Detection of the viral genome through RT-qPCR is the golden standard for this test, however, the high demand of the materials and reagents needed to sample individuals, purify the viral RNA, and perform the RT-qPCR test has resulted in a worldwide shortage of several of these supplies. Here, we show that directly lysed saliva samples can serve as a suitable source for viral RNA detection that is cheaper and can be as efficient as the classical protocol that involves column purification of the viral RNA. In addition, it surpasses the need for swab sampling, decreases the risk of the healthcare personnel involved in this process, and accelerates the diagnostic procedure.

Background:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is a current public health concern.Rapid diagnosis is crucial, and reverse transcription polymerase chain reaction (RT-PCR) is presently the reference standard for SARS-CoV-2 detection. Objective: Automated RT-PCR analysis (ARPA) is a software designed to analyze RT-PCR data for SARS-CoV-2 detection. ARPA loads the RT-PCR data, classifies each sample by assessing its amplification curve behavior, evaluates the experiment's quality, and generates reports. Methods: ARPA was implemented in the R language and deployed as a Shiny application. We evaluated the performance of ARPA in 140 samples. The samples were manually classified and automatically analyzed using ARPA. Results: ARPA had a true-positive rate = 1, true-negative rate = 0.98, positive-predictive value = 0.95, and negative-predictive value = 1, with 36 samples correctly classified as positive, 100 samples correctly classified as negative, and two samples classified as positive even when labeled as negative by manual inspection. Two samples were labeled as invalid by ARPA and were not considered in the performance metrics calculation. Conclusions: ARPA is a sensitive and specific software that facilitates the analysis of RT-PCR data, and its implementation can reduce the time required in the diagnostic pipeline. (REV INVEST CLIN. [AHEAD OF PRINT]

Rotaviruses have a genome composed of 11 segments of double-stranded RNA. Whether all 5′ ends of the positive-sense genomic RNAs contained in the mature viral particles are modified by a cap structure is unknown.