BACKGROUND:Data on the population-scale impact of dolutegravir (DTG)-based HIV regimens in sub-Saharan Africa are extremely limited. We used data from a surveillance cohort in southern Uganda to assess viral suppression and antiretroviral (ART) resistance over 10-years alongside DTG scale-up. METHODS:Consenting participants in the population-based Rakai Community Cohort Study between August 2011 and March 2023 aged 15-49 completed questionnaires and provided samples for HIV testing, viral load quantification, and viral deep-sequencing. We collected data on DTG utilization at HIV care clinics. We estimated the prevalence of HIV suppression and ART resistance using robust Poisson regression. Bayesian logistic regression quantified associations between resistance and individual-level suppression across surveys. RESULTS:Among 8781 people with HIV (PWH), suppression increased from 57.1% (2014, 95% confidence interval [CI], 55.4%-58.8%) to 90.3% (2022, 95% CI, 89.2%-91.4%). By 2020 84.4% (95% CI, 83.7%-85.2%) and 64.6% (95% CI, 63.9%-65.3%) of men and women on ART were on DTG. Among treatment-experienced viremic PWH, any intermediate/high resistance decreased from 51.1% (95% CI, 40.7%-64.2%, 2014) to 27.9% (95% CI, 21.3%-36.5%, 2022). Two of 258 (0.8%) 2022 participants harbored intermediate/high-level DTG resistance (inQ148R, inE138K, and inG140A). inS153Y (2-fold INSTI resistance) was observed in 23/306 (7.5%) of viremic individuals, with evidence of transmission. By 2022, NNRTI/NRTI resistance was not associated with a reduction in individual-level suppression (risk ratios: 1.15, 95% HPD: 0.93-1.39; 1.14, 0.86-1.42). CONCLUSIONS:Viral suppression increased during the DTG transition with minimal emerging intermediate/high-level resistance. Falling resistance among treatment-experienced PWH underscores the role of ART adherence in reducing viremia. The emergence of inS153Y justifies continued surveillance.

Broadly neutralizing antibodies (bnAbs), passively administered or elicited through vaccination, are a promising strategy for novel HIV prevention, treatment or inducing ART-free remission. However, HIV diversity and evolution are a barrier to the efficacy of bnAbs and there is therefore an urgent need for continuous virus surveillance to identify bnAbs with optimal neutralization breadth and potency against transmitted/founder (TF) viruses, especially in high-burden regions. We determined the neutralization sensitivity of TF viruses isolated within seven days after first detection of heterosexually acquired infection from young women 18-23 years old (n = 39) and within 1 month after birth from in-utero infected infants (n = 21) from FRESH and Baby Cure cohorts respectively, in KwaZulu-Natal, South Africa, where HIV-1 subtype C predominates. Neutralization sensitivities of 47 viruses from FRESH and 21 viruses from Baby Cure were assessed against nine bnAbs targeting different regions on the HIV-1 Env trimer. HIV-1 env sequences within and between bnAb epitopes were compared with database. The bnAbs VRC07-523LS, CAP256-VRC26.25, PGDM1400, 10E8 and PGT151 displayed higher neutralization breadth and potency than other bnAbs against FRESH TF viruses (>70% coverage, starting concentration of 10 μg/ml). Furthermore, VRC07-523LS showed higher neutralization breadth and potency than other bnAbs against Baby Cure TF viruses (p = 0.02). Interestingly, CAP256-VRC26.25 and PGT151 had lower neutralization coverage against infant TF viruses (<60% coverage). Moreover, 40% of infants TF had escape mutations within the V2 loop compared to 28% observed in FRESH and these mutations may explain the observed differences in neutralization sensitivities. However, few mutations were observed in gp120-gp41 interface in both adults and infants. Our findings suggest that intervention studies may have to consider different antibody combinations in adult versus paediatric settings. Moreover, high transmission of escape variants in both vertical and heterosexual transmissions is of concern. This information may be important in the selection of bnAbs that will undergo clinical testing in subtype C settings.

The National Collection of Type Cultures (NCTC) was founded on 1 January 1920 in order to fulfil a recognized need for a centralized repository for bacterial and fungal strains within the UK. It is among the longest-established collections of its kind anywhere in the world and today holds approximately 6000 type and reference bacterial strains – many of medical, scientific and veterinary importance – available to academic, health, food and veterinary institutions worldwide. Recently, a collaboration between NCTC, Pacific Biosciences and the Wellcome Sanger Institute established the NCTC3000 project to long-read sequence and assemble the genomes of up to 3000 NCTC strains. Here, at the beginning of the collection’s second century, we introduce the resulting NCTC3000 sequence read datasets, genome assemblies and annotations as a unique, historically and scientifically relevant resource for the benefit of the international bacterial research community.

Although certain individuals with HIV infection can stop antiretroviral therapy (ART) without viral load rebound, the mechanisms under-pinning ‘post-treatment control’ remain unclear. Using RNA-Seq we explored CD4 T cell gene expression to identify evidence of a mechanism that might underpin virological rebound and lead to discovery of associated biomarkers. Fourteen female participants who received 12 months of ART starting from primary HIV infection were sampled at the time of stopping therapy. Two analysis methods (Differential Gene Expression with Gene Set Enrichment Analysis, and Weighted Gene Co-expression Network Analysis) were employed to interrogate CD4+ T cell gene expression data and study pathways enriched in post-treatment controllers versus early rebounders. Using independent analysis tools, expression of genes associated with type I interferon responses were associated with a delayed time to viral rebound following treatment interruption (TI). Expression of four genes identified by Cox-Lasso ( ISG15 , XAF1 , TRIM25 and USP18 ) was converted to a Risk Score, which associated with rebound ( p < 0.01). These data link transcriptomic signatures associated with innate immunity with control following stopping ART. The results from this small sample need to be confirmed in larger trials, but could help define strategies for new therapies and identify new biomarkers for remission.

Enzymatic DNA modifications like methylcytosine (5mdC), methyladenine (N6mdA), or hydroxymethylcytosine (5hmdC) are key for chromatin function, gene expression regulation, and antiviral defense, but they remain understudied in non-model organisms. We established a mass spectrometric method for the sensitive and accurate quantification of enzymatic DNA modifications, and analyzed 85 bacterial genomes, 19 plant samples, 41 tissues from 12 animal species, 6 yeast species, and two archaeal species. We report no or only very low concentrations of DNA modifications in yeast and insects, but find DNA modifications universal to both bacteria and higher eukaryotes. Specifically for prokaryotes, our dataset indicates that evolutionary relationships and host–pathogen interactions, but not the ecological niche in general, select for a similar degree of DNA modification. In higher eukaryotes, largest concentration differences between tissues are detected for 5hmdC. Our dataset further reveals unique biological cases that warrant attention in the study of DNA modifications. For instance, while our data shows that most species contain just one dominating DNA modification, we detect all dominianting DNA modifications (5mdC, N6mdA, and 5hmdC) to exist in parallel in Raphanus sativus . Other plant species, like onion, sunflower, or the grass big bluestem, can have more than 35% of cytosines methylated. Finally, 5hmdC, so far mostly studied in the vertebrate central nervous system, was identified to reach a concentration of up to 8% of all cytosines in the Oman garra brain, and was also detected in several plants, like Lepidium sativum . The present study underscores the exploitation of biological diversity for studying DNA modifications.

Interpreting the function and metabolism of enzymatic DNA modifications requires both position-specific and global quantities. Sequencing-based techniques that deliver the former have become broadly accessible, but analytical methods for the global quantification of DNA modifications have thus far been applied mostly to individual problems. We established a mass spectrometric method for the sensitive and accurate quantification of multiple enzymatic DNA modifications. Then, we isolated DNA from 124 archean, bacterial, fungal, plant, and mammalian species, and several tissues and created a resource of global DNA modification quantities. Our dataset provides insights into the general nature of enzymatic DNA modifications, reveals unique biological cases, and provides complementary quantitative information to normalize and assess the accuracy of sequencing-based detection of DNA modifications. We report that only three of the studied DNA modifications, methylcytosine (5mdC), methyladenine (N6mdA) and hydroxymethylcytosine (5hmdC), were detected above a picomolar detection limit across species, and dominated in higher eukaryotes (5mdC), in bacteria (N6mdA), or the vertebrate central nervous systems (5hmdC). All three modifications were detected simultaneously in only one of the tested species, Raphanus sativus . In contrast, these modifications were either absent or detected only at trace quantities, across all yeasts and insect genomes studied. Further, we reveal interesting biological cases. For instance, in Allium cepa , Helianthus annuus , or Andropogon gerardi , more than 35% of cytosines were methylated. Additionally, next to the mammlian CNS, 5hmdC was also detected in plants like Lepidium sativum and was found on 8% of cytosines in the Garra barreimiae brain samples. Thus, identifying unexpected levels of DNA modifications in several wild species, our resource underscores the need to address biological diversity for studying DNA modifications.

Very early initiation of antiretroviral therapy (ART) in pediatric HIV infection offers a unique opportunity to limit the size and diversity of the viral reservoir. However, only rarely is ART alone sufficient to achieve remission.

Objective: The worldwide incidence of pregnancy for women living with perinatal HIV infection is increasing. Subsequently, there is growing risk of second-generation mother-to-child HIV transmission. The infant clinical outcomes for such a phenomenon have yet to be described. Design: As part of a wider observational study in KwaZulu-Natal, South Africa, six infants with in-utero HIV infection were identified as being born to mothers with perinatal HIV infection. Methods: Blood results and clinical data were collected in the first 3 years of life. In two cases, sample availability allowed confirmation by phylogenetic analysis of grandmother-to-mother-to-child HIV transmission. Results: Outcomes were poor in all six cases. All six mothers had difficulty administering twice daily combination antiretroviral therapy to their infants due to difficulties with acceptance, disclosure, poor health and being themselves long-term nonprogressors. Nonnucleoside reverse transcriptase inhibitor-resistant virus was detected in all mothers tested. None of the infants maintained suppression of viraemia on combination antiretroviral therapy. One infant died, and another was lost to follow-up. Conclusion: As the numbers of second-generation mother-to-child transmissions increase, it is important to highlight that this mother–infant dyad represents an extremely vulnerable group. In order for them to survive and thrive, these infants’ mothers require their specific needs to be addressed and given intensive support.

Introduction The lack of approved specific therapeutic agents to treat coronavirus disease (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to the rapid implementation of convalescent plasma therapy (CPT) trials in many countries, including the United Kingdom. Effective CPT is likely to require high titres of neutralising antibody (nAb) in convalescent donations. Understanding the relationship between functional neutralising antibodies and antibody levels to specific SARS-CoV-2 proteins in scalable assays will be crucial for the success of a large-scale collection. We assessed whether neutralising antibody titres correlated with reactivity in a range of enzyme-linked immunosorbent assays (ELISA) targeting the spike (S) protein, the main target for human immune response. Methods Blood samples were collected from 52 individuals with a previous laboratory-confirmed SARS-CoV-2 infection. These were assayed for SARS-CoV-2 nAbs by microneutralisation and pseudo-type assays and for antibodies by four different ELISAs. Receiver operating characteristic (ROC) analysis was used to further identify sensitivity and specificity of selected assays to identify samples containing high nAb levels. Results All samples contained SARS-CoV-2 antibodies, whereas neutralising antibody titres of greater than 1:20 were detected in 43 samples (83% of those tested) and >1:100 in 22 samples (42%). The best correlations were observed with EUROimmun immunoglobulin G (IgG) reactivity (Spearman Rho correlation coefficient 0.88; p < 0.001). Based on ROC analysis, EUROimmun would detect 60% of samples with titres of >1:100 with 100% specificity using a reactivity index of 9.1 (13/22). Discussion Robust associations between nAb titres and reactivity in several ELISA-based antibody tests demonstrate their possible utility for scaled-up production of convalescent plasma containing potentially therapeutic levels of anti-SARS-CoV-2 nAbs.

Background. The extent of spread of SARS coronavirus 2 (SARS-CoV-2) in the UK and elsewhere is unknown because typically only symptomatic individuals are diagnosed. We performed a serological study of recent blood donors in Scotland to detect antibodies to 45 SARS-CoV-2 as a marker of past infection. Methods. A pseudotyped SARS-CoV-2 virus microneutralisation assay was used to detect neutralising antibodies to SARS-CoV-2. The study group comprised samples from 1000 blood donors collected in Scotland during March, 2020. Controls were collected from 100 50 donors in Scotland during 2019. Findings. All samples collected on the 17 th March, 2020 (n=500) were negative in the pseudotyped SARS-CoV-2 virus microneutralisation assay. Neutralising antibodies were detected in 5 of the 500 samples collected 21 st – 23 rd March; one further sample was reactive 55 in an anti-spike ELISA. (Nab) titres were determined by endpoint two-fold serial dilutions of test samples mixed with 10 5 relative light units (RLU) of pseudotyped virus, incubated at 37°C for two hours and then mixed with 10 4 HEK 293T ACE2-transfected cells per well. Plates were incubated for 72 hours at 37°C and then cells were lysed and assayed for luciferase expression. Neutralization titres are expressed as (cid:1)(cid:2) (cid:3)(cid:4)

Background The progression and geographical distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the United Kingdom (UK) and elsewhere is unknown because typically only symptomatic individuals are diagnosed. We performed a serological study of blood donors in Scotland in the spring of 2020 to detect neutralising antibodies to SARS-CoV-2 as a marker of past infection and epidemic progression. Aim Our objective was to determine if sera from blood bank donors can be used to track the emergence and progression of the SARS-CoV-2 epidemic. Methods A pseudotyped SARS-CoV-2 virus microneutralisation assay was used to detect neutralising antibodies to SARS-CoV-2. The study comprised samples from 3,500 blood donors collected in Scotland between 17 March and 18 May 2020. Controls were collected from 100 donors in Scotland during 2019. Results All samples collected on 17 March 2020 (n = 500) were negative in the pseudotyped SARS-CoV-2 virus microneutralisation assay. Neutralising antibodies were detected in six of 500 donors from 23 to 26 March. The number of samples containing neutralising antibodies did not significantly rise after 5–6 April until the end of the study on 18 May. We found that infections were concentrated in certain postcodes, indicating that outbreaks of infection were extremely localised. In contrast, other areas remained comparatively untouched by the epidemic. Conclusion Although blood donors are not representative of the overall population, we demonstrated that serosurveys of blood banks can serve as a useful tool for tracking the emergence and progression of an epidemic such as the SARS-CoV-2 outbreak.

Background: Early combination antiretroviral therapy (cART) reduces the size of the viral reservoir in paediatric and adult HIV infection. Very early-treated children may have higher cure/remission potential. Methods: In an observational study of 151 in utero (IU)-infected infants in KwaZulu-Natal, South Africa, whose treatment adhered strictly to national guidelines, 76 infants diagnosed via point-of-care (PoC) testing initiated cART at a median of 26 h (IQR 18-38) and 75 infants diagnosed via standard-of-care (SoC) laboratory-based testing initiated cART at 10 days (IQR 8-13). We analysed mortality, time to suppression of viraemia, and maintenance of aviraemia over the first 2 years of life. Findings: Baseline plasma viral loads were low (median 8000 copies per mL), with 12% of infants having undetectable viraemia pre-cART initiation. However, barely one-third (37%) of children achieved suppression of viraemia by 6 months that was maintained to >12 months. 24% had died or were lost to follow up by 6 months. Infant mortality was 9.3%. The high-frequency virological failure in IU-infected infants was associated not with transmitted or acquired drug-resistant mutations but with cART non-adherence (plasma cART undetectable/subtherapeutic, p<0.0001) and with concurrent maternal cART failure (OR 15.0, 95%CI 5.6-39.6; p<0.0001). High-frequency virological failure was observed in PoC- and SoC-tested groups of children. Interpretation: The success of early infant testing and cART initiation strategies is severely limited by subsequent cART non-adherence in HIV-infected children. Although there are practical challenges to administering paediatric cART formulations, these are overcome by mothers who themselves are cART-adherent. These findings point to the ongoing obligation to address the unmet needs of the mothers. Eliminating the particular barriers preventing adequate treatment for these vulnerable women and infants need to be prioritised in order to achieve durable suppression of viraemia on cART, let alone HIV cure/remission, in HIV-infected children. (C) 2020 The Author(s). Published by Elsevier Ltd.

Previous studies of the transmitted/founder virus compared to viral quasispecies in the donor have yielded conflicting results. In heterosexual adult-to-adult transmission (ATAT), the viral replicative capacity (VRC) of transmitted virus is reportedly either similar to, or somewhat higher than, that of donor virus, whilst transmitted virus in mother-to-child transmission (MTCT) has a significantly lower VRC than that of maternal virus. These discrepancies may be explained by the different methodologies used in these studies, or they may reflect true differences in the transmission bottleneck. To resolve this question, we here use the same methodology to compare transmitted versus donor virus in MTCT and ATAT. We show that, in a South African mother-child cohort, infant virus samples obtained at 1-2 days after birth had VRC significantly lower than in the mothers (p=0.0003). By contrast, in Zambian ATAT transmission pairs, VRC of transmitted virus was similar to or somewhat higher than donor virus (p=ns). The VRC of virus transmitted to the recipient, compared to that in the donor, was significantly lower in MTCT versus heterosexual ATAT (p=0.01). These studies demonstrate that fundamental differences exist between the viruses transmitted via the MTCT and ATAT bottlenecks that are not explained by methodological factors. This result is of importance since transmission of low replicative capacity virus results in low immune activation and a small viral reservoir, and therefore the preferential transmission of low fitness viruses in MTCT might be expected to increase cure potential in in utero infected infants and children.IMPORTANCE Understanding the factors determining which viruses are preferentially transmitted in HIV infection is critical to the development of new, effective strategies to prevent transmission. Despite this, much remains unknown in this respect, both with regard to adult-to-adult transmission (ATAT) but especially with respect to mother-to-child transmission (MTCT). The finding here that fundamental differences exist in the genetic bottleneck of HIV transmission between heterosexual ATAT and MTCT is an important initial step to help define the viral mechanisms contributing to transmission in each case. In addition, we show that viruses of low viral replicative capacity are preferentially transmitted in MTCT. This suggests the possibility that a window of opportunity exists following in utero infection in which early anti-viral intervention not only reduces the size and diversity of the viral reservoir, but additionally maintains a reservoir comprising low viral replicative capacity HIV. Low replicative capacity of transmitted virus has previously been shown to result in low immune activation and low proviral DNA load in central memory cells, factors likely to be directly relevant to increasing cure potential in HIV-infected infants and children.

Raoultella terrigena is a bacterial species associated with soil and aquatic environments; however, sporadic cases of opportunistic disease in humans have been reported. Here, we report the first two complete genome sequences from clinical strains isolated from human sources that have been deposited in the National Collection of Type Cultures (NCTC).

Kocuria rosea is a Gram-positive coccus found in the environment and within normal human skin microbiota, and more recently, it has been potentially implicated as an opportunistic pathogen. Here, we describe the genome sequences of five strains of K. rosea (NCTC2676, NCTC7514, NCTC7512, NCTC7528, and NCTC7511).

Haemophilus haemolyticus is a Gram-negative bacterium that is a commensal of the respiratory tract in humans. Here, we report the complete genome sequence available for Haemophilus haemolyticus strain NCTC 10839, which was originally isolated from the nasopharynx of a child.

Background: Reports of posttreatment control following antiretroviral therapy (ART) have prompted the question of how common immune control of HIV infection is in the absence of ART. In contrast to adult infection, where elite controllers have been very well characterized and constitute approximately 0.5% of infections, very few data exist to address this question in paediatric infection. Methods: We describe 11 ART-naive elite controllers from 10 cohorts of HIV-infected children being followed in South Africa, Brazil, Thailand, and Europe. Results: All but one of the elite controllers (91%) are females. The median age at which control of viraemia was achieved was 6.5 years. Five of these 11 (46%) children lost control of viraemia at a median age of 12.9 years. Children who maintained control of viraemia had significantly higher absolute CD4(+) cell counts in the period of elite control than those who lost viraemic control. On the basis of data available from these cohorts, the prevalence of elite controllers in paediatric infection is estimated to be 5-10-fold lower than in adults. Conclusion: Although conclusions are limited by the study design, these data suggest that, whilst paediatric elite control can be achieved, compared with adult elite controllers, this occurs rarely, and takes some years after infection to achieve. Also, loss of immune control arises in a high proportion of children and often relatively rapidly. These findings are consistent with the more potent antiviral immune responses observed in adults and in females. Copyright (C) 2018 Wolters Kluwer Health, Inc. All rights reserved.

Epstein-Barr virus (EBV) replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of > 8,000 cellular and 69 EBV proteins, including > 500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcomaassociated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.

ABSTRACT Epstein-Barr virus (EBV) is a ubiquitous pathogen of humans that can cause several types of lymphoma and carcinoma. Like other herpesviruses, EBV has diversified through both coevolution with its host and genetic exchange between virus strains. Sequence analysis of the EBV genome is unusually challenging because of the large number and lengths of repeat regions within the virus. Here we describe the sequence assembly and analysis of the large internal repeat 1 of EBV (IR1; also known as the BamW repeats) for more than 70 strains. The diversity of the latency protein EBV nuclear antigen leader protein (EBNA-LP) resides predominantly within the exons downstream of IR1. The integrity of the putative BWRF1 open reading frame (ORF) is retained in over 80% of strains, and deletions truncating IR1 always spare BWRF1. Conserved regions include the IR1 latency promoter (Wp) and one zone upstream of and two within BWRF1. IR1 is heterogeneous in 70% of strains, and this heterogeneity arises from sequence exchange between strains as well as from spontaneous mutation, with interstrain recombination being more common in tumor-derived viruses. This genetic exchange often incorporates regions of <1 kb, and allelic gene conversion changes the frequency of small regions within the repeat but not close to the flanks. These observations suggest that IR1—and, by extension, EBV—diversifies through both recombination and breakpoint repair, while concerted evolution of IR1 is driven by gene conversion of small regions. Finally, the prototype EBV strain B95-8 contains four nonconsensus variants within a single IR1 repeat unit, including a stop codon in the EBNA-LP gene. Repairing IR1 improves EBNA-LP levels and the quality of transformation by the B95-8 bacterial artificial chromosome (BAC). IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the world population but causes illness in only a small minority of people. Nevertheless, over 1% of cancers worldwide are attributable to EBV. Recent sequencing projects investigating virus diversity to see if different strains have different disease impacts have excluded regions of repeating sequence, as they are more technically challenging. Here we analyze the sequence of the largest repeat in EBV (IR1). We first characterized the variations in protein sequences encoded across IR1. In studying variations within the repeat of each strain, we identified a mutation in the main laboratory strain of EBV that impairs virus function, and we suggest that tumor-associated viruses may be more likely to contain DNA mixed from two strains. The patterns of this mixing suggest that sequences can spread between strains (and also within the repeat) by copying sequence from another strain (or repeat unit) to repair DNA damage.