Detection of SARS-CoV-2 RNA in Bivalve Mollusks by Droplet Digital RT-PCR (dd RT-PCR)

Bivalve shellfish are readily contaminated by human pathogens present in waters impacted by municipal sewage, and the detection of SARS-CoV-2 in feces of infected patients and in wastewater has drawn attention to the possible presence of the virus in bivalves. The aim of this study was to collect data on SARS-CoV-2 prevalence in bivalve mollusks from harvesting areas of Campania region. A total of 179 samples were collected between September 2019 and April 2021 and were tested using droplet digital RT-PCR (dd RT-PCR) and real-time RT-PCR. Combining results obtained with different assays, SARS-CoV-2 presence was detected in 27/179 (15.1%) of samples. A median viral concentration of 1.1 × 102 and 1.4 × 102 g.c./g was obtained using either Orf1b nsp14 or RdRp/gene E, respectively. Positive results were unevenly distributed among harvesting areas and over time, positive samples being more frequent after January 2021. Partial sequencing of the spike region was achieved for five samples, one of which displaying mutations characteristic of the Alpha variant (lineage B.1.1.7). This study confirms that bivalve mollusks may bioaccumulate SARS-CoV-2 to detectable levels and that they may represent a valuable approach to track SARS-CoV-2 in water bodies and to monitor outbreak trends and viral diversity.

Discriminatory Weight of SNPs in Spike SARS-CoV-2 Variants: A Technically Rapid, Unambiguous, and Bioinformatically Validated Laboratory Approach

Background: The SARS-CoV-2 virus has assumed considerable importance during the COVID-19 pandemic. Its mutation rate is high, involving the spike (S) gene and thus there has been a rapid spread of new variants. Herein, we describe a rapid, easy, adaptable, and affordable workflow to uniquely identify all currently known variants through as few analyses. Our method only requires two conventional PCRs of the S gene and two Sanger sequencing reactions, and possibly another PCR/sequencing assay on a N gene portion to identify the B.1.160 lineage. Methods: We selected an S gene 1312 bp portion containing a set of SNPs useful for discriminating all variants. Mathematical, statistical, and bioinformatic analyses demonstrated that our choice allowed us to identify all variants even without looking for all related mutations, as some of them are shared by different variants (e.g., N501Y is found in the Alpha, Beta, and Gamma variants) whereas others, that are more informative, are unique (e.g., A57 distinctive to the Alpha variant). Results: A “weight” could be assigned to each mutation that may be present in the selected portion of the S gene. The method’s robustness was confirmed by analyzing 80 SARS-CoV-2-positive samples. Conclusions: Our workflow identified the variants without the need for whole-genome sequencing and with greater reliability than with commercial kits.

SARS-CoV-2 variants genome analysis of Indonesian isolates and their responses to available vaccines

Background SARS-CoV-2 is a virus that initially appeared in Wuhan, China, at the end of 2019. Since then, the virus has spread until to almost all countries resulting in a global pandemic. Over time, this virus continues to mutate and produce several other variants. In Indonesia, there are multiple variants of SARS-CoV-2 identified, as well as various local variants that are not yet considered to be ‘variants of concern’. Therefore, this investigation is intended to understand the prevalence and epidemiology of the virus, along with detecting the mutations that occur in genes associated with whole-genome-sequences (WGS) isolated in Indonesia. Result Analyses were performed to investigate SARS-CoV-2 prevalence in Indonesia using data obtained from GISAID.org. A whole-genome sequencing was performed on the random samples taken from GISAID.org utilizing the BLAST tool from NCBI. The variants identified in Indonesia are Alpha, Beta and Delta variants, as well as local variants B.1.470 and B.1.466.2. As of the end of November, it was found that there are a total of 5.348 cases of the Delta, 78 cases of the Alpha, 22 cases of the Beta, 572 cases of the local variant B.1.470, and 1.833 cases of the local variant B.1.466.2. Other cases include 219 cases of local variant B.1.1.398, 160 cases of local variant B.1.459 and 1.028 cases of the wild type. In total there are 9.260 isolated genomes collected in GISAID that are located in Indonesia. Using BLAST, WGS of Alpha, Delta, Beta, B.1.470 and B.1.466.2 variants isolated in Indonesia was compared with the wild type from Wuhan NC.045512.2. It was found that multiple mutations have occurred in the samples. The mutations identified as are H69del, V70I, N501Y, D614G, A570D, P681H, T716I, S982A, and D1118H in the Alpha variant, T19R, L452R, T478K, D614G, and D950N in the Delta variant, D215G, D614G, A701V, L241-, L242-, K417N in the Beta variant, D614G, L242F, and S12F in the B.1.470 variant and D614G, N439K, and P681R in the B.1.466.2 variant. These mutations had caused alterations in the characteristics of the virus and how it may affect vaccine efficacy. Conclusions The results from whole-genome sequencing of variants isolated in Indonesia have found that multiple mutations have occurred in genes of the SARS-CoV-2 Virus and it caused alterations in the characteristics of the virus and may affect vaccine efficacy. It should be noted that classification from the GISAID website may change overtime. The result in this paper is based on the data taken at the end of November.

Modelling COVID-19 Vaccine Breakthrough Infections in Highly Vaccinated Israel - the effects of waning immunity and third vaccination dose

In August 2021, a major wave of the SARS-CoV-2 Delta variant erupted in the highly vaccinated population of Israel. The Delta variant has a transmission advantage over the Alpha variant, and thus replaced it in approximately two months. The outbreak led to an unexpectedly large proportion of breakthrough infections (BTI)-- a phenomenon that received worldwide attention. The BTI proportion amongst cases in the age group of 60+ years reached levels as high as ~85% in August 2021. Most of the Israeli population, especially those 60+ age, received their second dose of the vaccination, four months before the invasion of the Delta variant. Hence, either the vaccine induced immunity dropped significantly or the Delta variant possesses immunity escaping abilities. In this work, we analyzed and model age-structured cases, vaccination coverage, and vaccine BTI data obtained from the Israeli Ministry of Health, to help understand the epidemiological factors involved in the outbreak. We propose a mathematical model which captures a multitude of factors, including age structure, the time varying vaccine efficacy, time varying transmission rate, BTIs, reduced susceptibility and infectivity of vaccinated individuals, protection duration of the vaccine induced immunity, and the vaccine distribution. We fitted our model to the cases among vaccinated and unvaccinated, for <60 and 60+ age groups, to address the aforementioned factors. We found that the transmission rate was driven by multiple factors including the invasion of Delta variant and the mitigation measures. Through a model reconstruction of the reproductive number R0(t), it was found that the peak transmission rate of the Delta variant was 1.96 times larger than the previous Alpha variant. The model estimated that the vaccine efficacy dropped significantly from >90% to ~40% over 6 months, and that the immunity protection duration has a peaked Gamma distribution (rather than exponential). We further performed model simulations quantifying the important role of the third vaccination booster dose in reducing the levels of breakthrough infections. This allowed us to explore "what if" scenarios should the booster not have been rolled out. Application of this framework upon invasion of new pathogens, or variants of concern, can help elucidate important factors in the outbreak dynamics and highlight potential routes of action to mitigate their spread.

Protective effect of a first SARS-CoV-2 infection from reinfection: a matched retrospective cohort study using PCR testing data in England

The duration of immunity after first SARS-CoV-2 infection and the extent to which prior immunity prevents reinfection is uncertain and remains an important question within the context of new variants. Using a retrospective population-based matched observational study approach, we identified cases with a first PCR positive test between 01 March 2020 and 30 September 2020 and cases were matched by age, sex, upper tier local authority of residence and testing route to individuals testing negative in the same week (controls) by PCR. After a 90-day pre-follow up period for cases and controls, any subsequent positive tests up to 31 December 2020 and deaths within 28 days of testing positive were identified, this encompassed an essentially vaccine-free period. There were 517,870 individuals in the matched cohort with 2,815 reinfection cases and 12,098 first infections. The protective effect of a prior SARS-CoV-2 PCR-positive episode was 78% (OR 0.22, 0.21-0.23). Protection rose to 82% (OR 0.18, 0.17-0.19) after a sensitivity analysis excluded 934 individuals with a first test between March and May and a subsequent positive test between June and September 2020. Amongst individuals testing positive by PCR during follow-up, reinfection cases had 77% lower odds of symptoms at the second episode (adjusted OR 0.23, 0.20-0.26) and 45% lower odds of dying in the 28 days after reinfection (adjusted OR 0.55, 0.42-0.71). Prior SARS-CoV-2 infection offered protection against reinfection in this population. There was some evidence that reinfections increased with the Alpha variant compared to the wild-type SARS-CoV-2 variant highlighting the importance of continued monitoring as new variants emerge.

The emergence, spread and vanishing of a French SARS-CoV-2 variant exemplifies the fate of RNA virus epidemics and obeys the Black Queen rule

The nature and dynamics of mutations associated with the emergence, spread and vanishing of SARS-CoV-2 variants causing successive waves are complex. We determined the kinetics of the most common French variant (Marseille-4) for 10 months since its onset in July 2020. Here, we analysed and classified into subvariants and lineages 7,453 genomes obtained by next-generation sequencing. We identified two subvariants, Marseille-4A, which contains 22 different lineages of at least 50 genomes, and Marseille-4B. Their average lifetime was 4.1+/-1.4 months, during which 4.1+/-2.6 mutations accumulated. Growth rate was 0.079+/-0.045, varying from 0.010 to 0.173. All the lineages exhibited a gamma distribution. Several beneficial mutations at unpredicted sites initiated a new outbreak, while the accumulation of other mutations resulted in more viral heterogenicity, increased diversity and vanishing of the lineages. Marseille-4B emerged when the other Marseille-4 lineages vanished. Its ORF8 gene was knocked out by a stop codon, as reported in several mink lineages and in the alpha variant. This subvariant was associated with increased hospitalization and death rates, suggesting that ORF8 is a nonvirulence gene. We speculate that the observed heterogenicity of a lineage may predict the end of the outbreak.

Is SARS-CoV-2 Omicron (B.1.1.529) variant causing different symptoms?

Background: Since preliminary evidence suggests that the new SARS-CoV-2 Omicron (B.1.1.529) variant may cause different symptoms and trigger outbreaks associated with less severe illness compared to previous strains, we conducted and infodemic analysis to verify these suppositions.Methods: We searched Google Trends using the most frequent COVID-19 symptoms, with “United Kingdom” country option and search periods “20-26 December 2020” (predominance of Alpha variant) and “19-25 December 2021” (Omicron prevalence >80%).Results: Seven symptoms (i.e., conjunctivitis, chills, cough, aches, fever, nausea and sore throat) appeared to be more searched in 2021 compared to 2020 (i.e., >15% increase), five symptoms (i.e., anosmia, tiredness, ageusia, sneezing and shortness of breath) were found to be less searched in 2021 compared to 2020 (i.e., >15% decrease), whilst the number of Google searches for headache, diarrhea and runny nose were almost comparable between the two periods (i.e., <15% variation).Conclusion: Actual predominance of Omicron (B.1.1.529) variant in UK is associated with higher number of Google searches for mild symptoms (conjunctivitis, chills, cough, aches and fever), accompanied by considerable lower interest for a severe clinical sign like shortness of breath, which characterizes lower respiratory tract infection.

New variants of SARS-CoV-2, vaccine immune response and the Brazilian reality

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a highly pathogenic β-coronavirus, is the etiologic agent of coronavirus disease 2019 (COVID-19), which gave rise to a difficult to control pandemic, especially in Brazil. Approximately 4,000 mutations have been identified in SARS-CoV-2, with the majority being redundant without having any biological effect on the virus. The aim of the present study was to objectively understand how new SARS-CoV-2 variants can affect vaccine response, in addition to highlighting the current situation in Brazil in the face of the pandemic and considering epidemiological and immunological aspects of COVID-19. The main protective correlate investigated in most vaccines is the neutralizing antibody titer induced by immunizing agents, observed in the pre-clinical phase in animals, whose action is to block the binding of the spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor, preventing infection. Up to the second half of 2021, the variants that are of greatest concern worldwide and require molecular surveillance are Alpha variant (or B.1.1.7 lineage), Beta (or B.1.351 lineage), Gamma (or P1 lineage) and Delta (or B.1.617.2 lineage). Brazil finds itself in a highly unfavorable scenario, with the circulation of variants of concern, mainly Gamma and Delta, with high fatality rates for COVID-19 and low vaccination rate. Given the still latent situation of the COVID-19 pandemic in Brazil, the lack of global planning for action strategies for non-pharmacological prevention measures, there is an imminent risk of the emergence of new variants due to the finding of susceptible hosts and the high proliferative rate of SARS-CoV-2. It is urgent to increase the genotyping of positive samples isolated from infected individuals, the speed of vaccination of the entire population and the unification of non pharmacological preventive measures throughout the country.

Optimization of multiplex PCR composition to screen for SARS-CoV-2 variants of concern

Background: The ongoing COVID-19 pandemic has led to the emergence of several variants of concern. To rapidly identify those variants, screening samples for whole-genome sequencing (WGS) prioritization could be performed. Objective: We optimized the polymerase chain reaction (PCR) screening method to identify the mutation in spike and ORF1a regions. Methods: We adopted primers targeting mutation in spike and ORF1a region from another study. We optimized the PCR screening method using kits readily available in Indonesia. Firstly, we compared N1 and N2 primers as internal positive control. We also compared GoTaq® 1-Step RT-qPCR System and Indonesia TFRIC-19 BioCOV-19 for the multiplex reaction. We used the optimized composition to screen SARS-CoV-2 positive samples from April – June 2021. Samples with spike and/or ORF1a target failure were subjected to whole genome sequencing (WGS). Results: The results demonstrated the N2 BioCOV-19 reaction as the optimized multiplex PCR composition for spike and ORF1a mutations screening. Whole-genome sequencing has shown that a sample with spike and ORF1a targets failure to be Alpha variant, while other samples with single target failure as non-variants of concern. Therefore, a multiplex RT-PCR composition has been optimized to detect mutation in spike and ORF1a regions. Conclusion: We have optimized a multiplex RT-PCR composition to detect mutation in spike and ORF1a regions.

A Rare Case of 3:1 Alpha Variant

Since the first documentation of slow alpha variants in Goodwin et al., there has been a single case report with an actual electroencephalography (EEG). However, any further case has not been reported since then, and neurologists are still unfamiliar with its presence due to its scarcity. Here, we present a rare case of 3:1 subharmonic alpha variant in a hope to acquaint EEG interpretations and speculate upon its benign nature.