The BNT162b2 mRNA vaccine is highly effective against SARS-CoV-2. However, apprehension exists that variants of concern (VOCs) may evade vaccine protection, due to evidence of reduced neutralization of the VOCs B.1.1.7 and B.1.351 by vaccine sera in laboratory assays. We performed a matched cohort study to examine the distribution of VOCs in infections of BNT162b2 mRNA vaccinees from Clalit Health Services (Israel) using viral genomic sequencing, and hypothesized that if vaccine effectiveness against a VOC is reduced, its proportion among breakthrough cases would be higher than in unvaccinated controls. Analyzing 813 viral genome sequences from nasopharyngeal swabs, we showed that vaccinees who tested positive at least 7 days after the second dose were disproportionally infected with B.1.351, compared with controls. Those who tested positive between 2 weeks after the first dose and 6 days after the second dose were disproportionally infected by B.1.1.7. These findings suggest reduced vaccine effectiveness against both VOCs within particular time windows. Our results emphasize the importance of rigorously tracking viral variants, and of increasing vaccination to prevent the spread of VOCs.

In some immunocompromised patients with chronic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, considerable adaptive evolution occurs. Some substitutions found in chronic infections are lineage-defining mutations in variants of concern (VOCs), which has led to the hypothesis that VOCs emerged from chronic infections. In this study, we searched for drivers of VOC-like emergence by consolidating sequencing results from a set of 27 chronic infections. Most substitutions in this set reflected lineage-defining VOC mutations; however, a subset of mutations associated with successful global transmission was absent from chronic infections. We further tested the ability to associate antibody evasion mutations with patient-specific and virus-specific features and found that viral rebound is strongly correlated with the emergence of antibody evasion. We found evidence for dynamic polymorphic viral populations in most patients, suggesting that a compromised immune system selects for antibody evasion in particular niches in a patient’s body. We suggest that a tradeoff exists between antibody evasion and transmissibility and that extensive monitoring of chronic infections is necessary to further understanding of VOC emergence.

The SARS-CoV-2 pandemic has been raging for over a year, creating global detrimental impact. The BNT162b2 mRNA vaccine has demonstrated high protection levels, yet apprehension exists that several variants of concerns (VOCs) can surmount the immune defenses generated by the vaccines. Neutralization assays have revealed some reduction in neutralization of VOCs B.1.1.7 and B.1.351, but the relevance of these assays in real life remains unclear. Here, we performed a case-control study that examined whether BNT162b2 vaccinees with documented SARS-CoV-2 infection were more likely to become infected with B.1.1.7 or B.1.351 compared with unvaccinated individuals. Vaccinees infected at least a week after the second dose were disproportionally infected with B.1.351 (odds ratio of 8:1). Those infected between two weeks after the first dose and one week after the second dose, were disproportionally infected by B.1.1.7 (odds ratio of 26:10), suggesting reduced vaccine effectiveness against both VOCs under different dosage/timing conditions. Nevertheless, the B.1.351 incidence in Israel to-date remains low and vaccine effectiveness remains high against B.1.1.7, among those fully vaccinated. These results overall suggest that vaccine breakthrough infection is more frequent with both VOCs, yet a combination of mass-vaccination with two doses coupled with non-pharmaceutical interventions control and contain their spread.

In some immunocompromised patients with chronic SARS-CoV-2 infection, dramatic adaptive evolution occurs, with substitutions reminiscent of those in variants of concern (VOCs). Here, we searched for drivers of VOC-like emergence by consolidating sequencing results from a set of twenty-seven chronic infections. Most substitutions in this set reflected lineage-defining VOC mutations, yet a subset of mutations associated with successful global transmission was absent from chronic infections. The emergence of these mutations might dictate when variants from chronic infections can dramatically spread onwards. Next, we tested the ability to predict antibody-evasion mutations from patient- and viral-specific features, and found that viral rebound is strongly associated with the emergence of antibody-evasion. We found evidence for dynamic polymorphic viral populations in most patients, suggesting that a compromised immune system selects for antibody-evasion in particular niches in a patient's body. We suggest that a trade-off exists between antibody-evasion and transmissibility that potentially constrains VOC emergence, and that monitoring chronic infections may be a means to predict future VOCs.

Genetic diversity is the fuel of evolution and facilitates adaptation to novel environments. However, our understanding of what drives differences in the genetic diversity during the early stages of viral infection is somewhat limited. Here, we use ultra-deep sequencing to interrogate 43 clinical samples taken from early infections of the human-infecting viruses HIV, RSV and CMV. Hundreds to thousands of virus templates were sequenced per sample, allowing us to reveal dramatic differences in within-host genetic diversity among virus populations. We found that increased diversity was mostly driven by presence of multiple divergent genotypes in HIV and CMV samples, which we suggest reflect multiple transmitted/founder viruses. Conversely, we detected an abundance of low frequency hyper-edited genomes in RSV samples, presumably reflecting defective virus genomes (DVGs). We suggest that RSV is characterized by higher levels of cellular co-infection, which allow for complementation and hence elevated levels of DVGs.

Background & aimsAcute hepatitis C (AHC) is not frequently identified because patients are usually asymptomatic, although may be recognized after iatrogenic exposures such as needle stick injuries, medical injection, and acupuncture. We describe an outbreak of AHC among 12 patients who received IV saline flush from a single multi-dose vial after intravenous contrast administration for a computerized tomography (CT) scan. The last patient to receive IV contrast with saline flush from a multi-dose vial at the clinic on the previous day was known to have chronic HCV genotype 1b (termed potential source, PS). Here we sought to confirm (via genetic analysis) the source of infection and to predict the minimal contaminating level of IV saline flush needed to transmit infectious virus to all patients.MethodsIn order to confirm the source of infection, we sequenced the HCV E1E2 region in 7 CT patients, in PS, and in 2 control samples from unrelated patients also infected with HCV genotype 1b. A transmission probabilistic model was developed to predict the contamination volume of blood that would have been sufficient to transmit infectious virus to all patients.ResultsViral sequencing showed close clustering of the cases with the PS. The transmission probabilistic model predicted that contamination of the multi-dose saline vial with 0.6–8.7 microliters of blood would have been sufficient to transmit infectious virus to all patients.ConclusionAnalysis of this unique cohort provides a new understanding of HCV transmission with respect to contaminating volumes and viral titers.

Obliterative bronchiolitis (OB) is the main cause of late mortality after lung transplantation. Cytomegalovirus infection has been associated with late graft failure. The aim of this study was to determine whether the development of OB was related to CMV pretransplant serological status and to CMV infections. The study group comprised 36 lung transplant recipients (27 HLT and 9 DLT) who survived more than 4 months, of whom 47% developed OB (defined by the persistence of an unexplained obstructive disease: FEV1/VC < 0,7). OB occurred more frequently: (1) in seronegative recipients with seropositive donors (8/9) than in seropositive recipients (7/19) or seronegative well‐matched recipients (2/8); and (2) in patients who experienced CMV pneumonia (11/16) and CMV recurrence (11/16). Since matching seronegative recipients is the best way to prevent CMV infection, we believe that seronegative grafts must be reserved for seronegative recipients.

1Mutations fuel evolution and facilitate adaptation to novel environments. However, 2 characterizing the spectrum of mutations in a population is obscured by high error rates of next 3 generation sequencing. Here, we present AccuNGS, a novel in vivo sequencing approach that 4 detects variants as rare as 1:10,000. Applying it to 46 clinical samples taken from early infections 5 of the human-infecting viruses HIV, RSV and CMV, revealed large differences in within-host 6 genetic diversity among virus populations. Haplotype reconstruction revealed that increased 7 diversity was mostly driven by multiple transmitted/founder viruses in HIV and CMV samples. 8Conversely, we detected an abundance of defective virus genomes (DVGs) in RSV samples, 9including hyper-edited genomes, nonsense mutations and single point deletions. Higher 10 proportions of DVGs correlated with increased viral loads, suggesting increased cellular co-11 infection rates, which enable DVG persistence. AccuNGS establishes a general platform that 12 allows detecting DVGs, and in general, rare variants that drive evolution. 13 2016; Wang, et al. 2017); error reduction by overlapping paired reads in paired-end sequencing 46 (Chen-Harris, et al. 2013;Schirmer, et al. 2015;Preston, et al. 2016); and usage of improved 47 polymerases (Imashimizu, et al. 2013). However, most experimental methods described above 48 are designed for samples with high biomass and are inapplicable for sequencing of clinical 49 samples, where the biomass may be extremely low. Furthermore, these experimental protocols 50 may introduce their own artifacts to the sequencing process (Lou, et al. 2013; Brodin, et al. 51 2015). On the computational side, it has been suggested that well-established variant callers do 52 not perform well on clinical virus samples (McCrone and Lauring 2016). Here, we sought to 53 develop a simple and rapid approach that can tackle the problem of accurate sequencing of 54 clinical samples, and applied it to study the early stages of virus infection. 55We describe AccuNGS, a simple yet powerful approach for accurate population sequencing and 56 bioinformatics variant calling. We extensively optimize all stages of the method to ensure high 57 accuracy and maximal yield. We use AccuNGS to perform in-depth sequencing of 46 samples 58 from three different major human pathogenic viruses: human immunodeficiency virus (HIV), 59 respiratory syncytial virus (RSV), and cytomegalovirus (CMV), all sampled during the acute 60 infection stage. We compare the within-host genetic diversity among and within different virus 61 populations, and find patterns characteristic of each virus. We demonstrate the role of multiple 62 transmitted/founder viruses as major contributors to the genetic diversity in HIV and CMV. 63 Furthermore, we identify and quantify the impact of various host editing enzymes on the 64 mutational spectrum of viral genomes/populations in vivo. Intriguingly, we find that RSV 65 samples bear much higher levels of potentially defective virus genome (DVGs) ...