Lineage replacement and evolution captured by the United Kingdom Covid Infection Survey

The Office for National Statistics COVID-19 Infection Survey is a large household-based surveillance study based in the United Kingdom. Here, we report on the epidemiological and evolutionary dynamics of SARS-CoV-2 determined by analysing sequenced samples collected up until 13th November 2021. We observed four distinct sweeps or partial-sweeps, by lineages B.1.177, B.1.1.7/Alpha, B.1.617.2/Delta, and finally AY.4.2, a sublineage of B.1.617.2, with each sweeping lineage having a distinct growth advantage compared to their predecessors. Evolution was characterised by steady rates of evolution and increasing diversity within lineages, but with step increases in divergence associated with each sweeping major lineage, leading to a faster overall rate of evolution and fluctuating levels of diversity. These observations highlight the value of viral sequencing integrated into community surveillance studies to monitor the viral epidemiology and evolution of SARS-CoV-2, and potentially other pathogens, particularly as routine PCR testing is phased out or in settings where large-scale sequencing is not feasible.

Nipah virus detection at bat roosts following spillover events in Bangladesh, 2012-2019

Knowledge of the dynamics and genetic diversity of Nipah virus circulating in bats and at the human-animal interface is limited by current sampling efforts, which produce few detections of viral RNA. We report on a series of investigations at bat roosts identified near human Nipah cases in Bangladesh between 2012 and 2019. Pooled bat urine samples were collected from 23 roosts; seven roosts (30%) had at least one sample with Nipah RNA detected from the first visit. In subsequent visits to these seven roosts, RNA was detected in bat urine up to 52 days after the presumed exposure of the human case, although the probability of detection declined rapidly with time. These results suggest that rapidly deployed investigations of Nipah virus shedding from bat roosts near human cases could increase the success of viral sequencing compared to background surveillance and enhance our understanding of Nipah virus ecology and evolution.

Molecular Epidemiology of Rabies in Wild Canidae in Tunisia

Rabies is a viral zoonosis that is transmissible to humans via domestic and wild animals. There are two epidemiological cycles for rabies, the urban and the sylvatic cycles. In an attempt to study the epidemiological role of wild canidae in rabies transmission, the present study aimed to analyze the genetic characteristics of virus isolates and confirm prior suggestions that rabies is maintained through a dog reservoir in Tunisia. Virus strains isolated from wild canidae were subject to viral sequencing, and Bayesian phylogenetic analysis was performed using Beast2 software. Essentially, the virus strains isolated from wild canidae belonged to the Africa-1 clade, which clearly diverges from fox-related strains. Our study also demonstrated that genetic characteristics of the virus isolates were not as distinct as could be expected if a wild reservoir had already existed. On the contrary, the geographic landscape is responsible for the genetic diversity of the virus. The landscape itself could have also acted as a natural barrier to the spread of the virus.

Transmission event of SARS-CoV-2 delta variant reveals multiple vaccine breakthrough infections

Background This study aims to identify the causative strain of SARS-CoV-2 in a cluster of vaccine breakthroughs. Vaccine breakthrough by a highly transmissible SARS-CoV-2 strain is a risk to global public health. Methods Nasopharyngeal swabs from suspected vaccine breakthrough cases were tested for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) by qPCR (quantitative polymerase chain reaction) for Wuhan-Hu1 and alpha variant. Positive samples were then sequenced by Swift Normalase Amplicon Panels to determine the causal variant. GATK (genome analysis toolkit) variants were filtered with allele fraction ≥80 and min read depth 30x. Results Viral sequencing revealed an infection cluster of 6 vaccinated patients infected with the delta (B.1.617.2) SARS-CoV-2 variant. With no history of vaccine breakthrough, this suggests the delta variant may possess immune evasion in patients that received the Pfizer BNT162b2, Moderna mRNA-1273, and Covaxin BBV152. Conclusions Delta variant may pose the highest risk out of any currently circulating SARS-CoV-2 variants, with previously described increased transmissibility over alpha variant and now, possible vaccine breakthrough. Funding Parts of this work was supported by the National Institute of Allergy and Infectious Diseases (1U19AI144297) and Baylor College of Medicine internal funding.

Rapid reinfection with SARS-CoV-2 variant-of-concern Alpha detected in a nurse during an outbreak at a non-covid inpatient ward: lessons learned

We describe the lessons learned during a SARS-CoV-2 variant-of-concern Alpha outbreak investigation at a normal care unit in a university hospital in Amsterdam in December 2020. The outbreak consisted of nine nurses and two roomed-in patient family members. (attack rate 18%). One nurse tested positive with a phylogenetically distinct variant, after a documented infection 83 days prior. Three key points were taken from this investigation. First, it was controlled by adherence to existing guidelines, despite increased transmissibility of the variant. Second, viral sequencing can inform transmission cluster inference, but the epidemiological context is essential to draw appropriate conclusions. Third, reinfections with Alpha variants can occur rapidly after primary infection.

Virus reactivation in a non-cirrhotic HBV patient requiring liver transplantation after cessation of nucleoside analogue therapy

Nucleos(t)ide analogues (NAs) are a mainstay of therapy for chronic hepatitis B (CHB) infections and have a profound effect on hepatitis B virus (HBV) suppression. We report a rare case of HBV reactivation in a CHB patient without cirrhosis following cessation of NA therapy that resulted in acute liver failure requiring liver transplantation. Investigation of the viral genetics and host immune responses suggest that viral mutations known to promote virus replication are associated with reactivation, whereas adaptive immunity to HBV remained defective in this patient. Viral sequencing may be useful for identifying mutations that are unfavorable for therapy withdrawal.

Immune profile and responses of a novel Dengue DNA vaccine encoding EDIII-NS1 consensus design based on Indo-African sequences

Following the recent clinical clearance of an Indian DNA COVID-19 vaccine, India and Africa are potential regions where DNA vaccines may become a major delivery system subject to a range of immunological and regulatory scrutiny. The ongoing COVID pandemic highlights the need to tackle viral variants and expand the number of antigens and assess diverse delivery systems. To address some of these key issues, we have created a Dengue DNA vaccine candidate with the EDIII region as the key antigen given the promise of this segment in not causing ADE, a challenge with this disease. In addition, we have added the NS1 region to broaden the immune response. Following a large Dengue viral sequencing exercise in India, complemented with data from east Africa, our approach was to generate a consensus of four serotypes ED3-NS1 vaccine to explore tackling the issue of diversity. Our In silico structural analysis of EDIII consensus vaccine sequence revealed that epitopes are structurally conserved and immunogenic across HLA diversity. Vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Furthermore, the DNA vaccination confers protection against DENV challenge in AG129 mice. Finally, assaying of intracellular staining for IFN-γ, immunoglobulin IgG2(a/c) /IgG1 ratios as well as immune gene profiling suggested a strong Th1-dominant immune response. Our Dengue DNA platform with a focus on Indo-African sequences offers an approach for assessing cross reactive immunity in animal models and lays the foundation for human vaccine roll out either as a stand-alone or mix and match strategy.

A Two-tiered Functional Screen Identifies Herpesviral Transcriptional Modifiers and their Essential Domains

While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel. Here, we used this approach to study the human oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV). We designed a sgRNA library containing all possible ~22,000 guides targeting the genome of KSHV - one cut site approximately every 8 base pairs - enabling the pooled screening of the entire genome. We used this tool to phenotype all possible Cas9-targeted viruses for transcription of KSHV late genes, which is required to produce structural components of the viral capsid. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we discovered a novel hit, ORF46 - and more specifically its DNA binding domain - is required for viral DNA replication. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.

Real-time monitoring and analysis of SARS-CoV-2 nanopore sequencing with minoTour.

Motivation: The ongoing SARS-CoV-2 pandemic has demonstrated the utility of real-time analysis of sequencing data, with a wide range of databases and resources for analysis now available. Here we show how the real-time nature of Oxford Nanopore Technologies sequencers can accelerate consensus generation, lineage and variant status assignment. We exploit the fact that multiplexed viral sequencing libraries quickly generate sufficient data for the majority of samples, with diminishing returns on remaining samples as the sequencing run progresses. We demonstrate methods to determine when a sequencing run has passed this point in order to reduce the time required and cost of sequencing. Results: We extended MinoTour, our real-time analysis and monitoring platform for nanopore sequencers, to provide SARS-CoV2 analysis using ARTIC network pipelines. We additionally developed an algorithm to predict which samples will achieve sufficient coverage, automatically running the ARTIC medaka informatics pipeline once specific coverage thresholds have been reached on these samples. After testing on run data, we find significant run time savings are possible, enabling flow cells to be used more efficiently and enabling higher throughput data analysis. The resultant consensus genomes are assigned both PANGO lineage and variant status as defined by Public Health England. Samples from within individual runs are used to generate phylogenetic trees incorporating optional background samples as well as summaries of individual SNPs. As minoTour uses ARTIC pipelines, new primer schemes and pathogens can be added to allow minoTour to aid in real-time analysis of pathogens in the future.

Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection Cases Corroborated by Sequencing

Evaluating the reinfection may offer some insight into areas for further investigation regarding durability of immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sixty cases of reinfection with viral sequencing were identified in PubMed, Embase, Web of Science, and medRxiv before May 1, 2021.Episodes of infection were separated by a median of 116 days. Severity of illness was greater among individuals reinfected within 90 days of initial infection, no asymptomatic initial cases developed severe reinfection, nearly half of cases had suspected escape variants, and nearly all individual tested following reinfection were found to have detectable levels of anti-SARS-CoV-2 antibodies. This analysis is limited by the heterogeneous methods used among reports. Reinfection continues to be relatively rare. As the case rate presumably increases over time, this review will inform measurements to determine the natural history and causal determinants of reinfection in more rigorous observational cohort studies and other standardized surveillance approaches.