Mature Rotavirus Particles Contain Equivalent Amounts of 7meGpppG-Capped and Noncapped Viral Positive-Sense RNAs.

Viruses have evolved different strategies to overcome their recognition by the host innate immune system. The addition of caps at their 5' RNA ends is an efficient mechanism not only to ensure escape from detection by the innate immune system but also to ensure the efficient synthesis of viral proteins. Rotavirus mRNAs contain a type 1 cap structure at their 5' end that is added by the viral capping enzyme VP3, which is a multifunctional protein with all the enzymatic activities necessary to add the cap and also functions as an antagonist of the 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway. Here, the relative abundances of capped and noncapped viral RNAs during the replication cycle of rotavirus were determined. We found that both classes of rotaviral plus-sense RNAs (+RNAs) were encapsidated and that they were present in a 1:1 ratio in the mature infectious particles. The capping of viral +RNAs was dynamic, since different ratios of capped and noncapped RNAs were detected at different times postinfection. Similarly, when the relative amounts of capped and uncapped viral +RNAs produced in an in vitro transcription system were determined, we found that the proportions were very similar to those in the mature viral particles and in infected cells, suggesting that the capping efficiency of VP3, both in vivo and in vitro, might be close to 50%. Unexpectedly, when the effect of simultaneously knocking down the expression of VP3 and RNase L on the cap status of viral +RNAs was evaluated, we found that, even though at late times postinfection there was an increased proportion of capped viral RNAs in infected cells, the viral particles isolated from this condition contained equal ratios of capped and noncapped viral RNA, suggesting that there might be selective packaging of capped and noncapped RNAs. IMPORTANCE 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. In this work, we characterized the relative proportions of capped and noncapped viral RNAs in rotavirus-infected cells and in viral particles by using a direct quantitative assay. We found that, independent of the relative proportions of capped/noncapped RNAs present in rotavirus-infected cells, there were similar proportions of these two kinds of 5'-modified positive-sense RNAs in the viral particles.

Automated Reverse Transcription Polymerase Chain Reaction Data Analysis for Sars-CoV-2 Detection.

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 SARSCoV-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.

Saliva Sampling and Its Direct Lysis, an Excellent Option To Increase the Number of SARS-CoV-2 Diagnostic Tests in Settings with Supply Shortages.

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 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Detection of the viral genome through reverse transcription-quantitative PCR (RT-qPCR) is the gold standard for this virus; however, the high demand of the materials and reagents needed to sample individuals, purify the viral RNA, and perform the RT-qPCR 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 less expensive and can be as efficient as the classical protocol, which involves column purification of the viral RNA. In addition, it bypasses the need for swab sampling, decreases the risk of the health care personnel involved in the testing process, and accelerates the diagnostic procedure.

Saliva sampling and its direct lysis is an excellent option for SARS-CoV-2 diagnosis in paediatric patients: comparison with the PanBio COVID-19 antigen rapid test in symptomatic and asymptomatic children.

Introduction. Lateral flow test (LFTs) have been used as an alternative to reverse transcription quantitative PCR (RT-qPCR) in point-of-care testing. Despite their benefits, the sensitivity of LFTs may be low and is affected by several factors. We have previously reported the feasibility of using direct lysis of individual or pools of saliva samples from symptomatic and asymptomatic patients as a source of viral genomes for detection by RT-qPCR.Hypothesis. Direct lysed saliva is more sensitive than antigen tests to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in samples from children.Aim. Our goals here were to valuate the specificity and sensitivity of the PanBio COVID-19 antigen rapid test device (Ag-RTD) compared with RT-qPCR of direct lysed saliva.Methodology. We evaluated the performance of the PanBio COVID-19 Ag-RTD in comparison to RT-qPCR direct lysed saliva from paired samples of 256 symptomatic and 242 asymptomatic paediatric patients.Results. Overall, although there were no differences in the specificity (96.6%), we found a lower sensitivity (64.3%) of the PanBio Ag-test RTD compared to saliva in both symptomatic and asymptomatic patients. In addition, the sensitivity of PanBio was not correlated with the viral load present in the samples.Conclusion. Our data highlight the benefits of using RT-qPCR and saliva samples for SARS-CoV-2 detection, particularly in paediatric patients.

Pooling saliva samples as an excellent option to increase the surveillance for SARS-CoV-2 when re-opening community settings.

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.

The Alpha Variant (B.1.1.7) of SARS-CoV-2 Failed to Become Dominant in Mexico.

During the coronavirus disease 2019 (COVID-19) pandemic, the emergence and rapid increase of the B.1.1.7 (Alpha) lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first identified in the United Kingdom in September 2020, was well documented in different areas of the world and became a global public health concern because of its increased transmissibility. The B.1.1.7 lineage was first detected in Mexico during December 2020, showing a slow progressive increase in its circulation frequency, which reached its maximum in May 2021 but never became predominant. In this work, we analyzed the patterns of diversity and distribution of this lineage in Mexico using phylogenetic and haplotype network analyses. Despite the reported increase in transmissibility of the B.1.1.7 lineage, in most Mexican states, it did not displace cocirculating lineages, such as B.1.1.519, which dominated the country from February to May 2021. Our results show that the states with the highest prevalence of B.1.1.7 were those at the Mexico-U.S. border. An apparent pattern of dispersion of this lineage from the northern states of Mexico toward the center or the southeast was observed in the largest transmission chains, indicating possible independent introduction events from the United States. However, other entry points cannot be excluded, as shown by multiple introduction events. Local transmission led to a few successful haplotypes with a localized distribution and specific mutations indicating sustained community transmission. IMPORTANCE 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. In this work, we analyzed the distribution of B.1.1.7 in Mexico using phylogenetic and haplotype network analyses. Our results show that the states with the highest prevalence of B.1.1.7 (around 30%) were those at the Mexico-U.S. border, which also exhibited the highest lineage diversity, indicating possible introduction events from the United States. Also, several haplotypes were identified with a localized distribution and specific mutations, indicating that sustained community transmission occurred in the country.

Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection.

Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe disease and are a significant burden to public health. Alphaviral replication results in the production of both capped and noncapped viral genomic RNAs (ncgRNAs), which are packaged into virions during infections of vertebrate and invertebrate cells. However, the roles that the ncgRNAs play during alphaviral infection have yet to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral infection was assessed by using mutations of the nsP1 protein of Sindbis virus (SINV), which altered the synthesis of the ncgRNAs during infection by modulating the protein's capping efficiency. Specifically, point mutations at residues Y286A and N376A decreased capping efficiency whereas a point mutation at D355A increased the capping efficiency of the SINV genomic RNA during genuine viral infection. Viral growth kinetics levels were significantly reduced for the D355A mutant relative to wild-type infection, whereas the Y286A and N376A mutants showed modest decreases in growth kinetics. Overall genomic translation and nonstructural protein accumulation were found to correlate with increases and decreases in capping efficiency. However, genomic, minus-strand, and subgenomic viral RNA synthesis were largely unaffected by the modulation of alphaviral capping activity. In addition, translation of the subgenomic alphaviral RNA (vRNA) was found not to be impacted by changes in capping efficiency. The mechanism by which the decreased presence of ncgRNAs reduced viral growth kinetics levels operated through the impaired production of viral particles. Collectively, these data illustrate the importance of ncgRNAs to viral infection and suggest that they play an integral role in the production of viral progeny.IMPORTANCE 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.