Search

Background We report outcomes and novel characterization of a unique cohort of 42 individuals with persistently indeterminate human immunodeficiency virus (HIV) status, the majority of whom are HIV viral controllers. Methods Eligible individuals had indeterminate or positive HIV serology, but persistently undetectable HIV ribonucleic acid (RNA) by commercial assays and were not taking antiretroviral therapy (ART). Routine investigations included HIV Western blot, HIV viral load, qualitative HIV-1 deoxyribonucleic acid (DNA), coinfection screen, and T-cell quantification. Research assays included T-cell activation, ART measurement, single-copy assays detecting HIV-1 RNA and DNA, and plasma cytokine quantification. Human immunodeficiency virus seropositivity was defined as ≥3 bands on Western blot; molecular positivity was defined as detection of HIV RNA or DNA. Results Human immunodeficiency virus infection was excluded in 10 of 42 referrals, remained unconfirmed in 2 of 42, and was confirmed in 30 of 42, who were identified as HIV elite controllers (ECs), normal CD4 T-cell counts (median 820/mL, range 805–1336), and normal CD4/CD8 ratio (median 1.8, range 1.2–1.9). Elite controllers had a median duration of elite control of 6 years (interquartile range = 4–14). Antiretroviral therapy was undetected in all 23 subjects tested. Two distinct categories of ECs were identified: molecular positive (n = 20) and molecular negative (n = 10). Conclusions Human immunodeficiency virus status was resolved for 95% of referrals with the majority diagnosed as EC. The clinical significance of the 2 molecular categories among ECs requires further investigation.

BACKGROUND: Despite circumstantial evidence for aerosol and fomite spread of SARS-CoV-2, empirical data linking either pathway with transmission are scarce. Here we aimed to assess whether the presence of SARS-CoV-2 on frequently-touched surfaces and residents' hands was a predictor of SARS-CoV-2 household transmission. METHODS: In this longitudinal cohort study, during the pre-alpha (September to December, 2020) and alpha (B.1.1.7; December, 2020, to April, 2021) SARS-CoV-2 variant waves, we prospectively recruited contacts from households exposed to newly diagnosed COVID-19 primary cases, in London, UK. To maximally capture transmission events, contacts were recruited regardless of symptom status and serially tested for SARS-CoV-2 infection by RT-PCR on upper respiratory tract (URT) samples and, in a subcohort, by serial serology. Contacts' hands, primary cases' hands, and frequently-touched surface-samples from communal areas were tested for SARS-CoV-2 RNA. SARS-CoV-2 URT isolates from 25 primary case-contact pairs underwent whole-genome sequencing (WGS). FINDINGS: From Aug 1, 2020, until March 31, 2021, 620 contacts of PCR-confirmed SARS-CoV-2-infected primary cases were recruited. 414 household contacts (from 279 households) with available serial URT PCR results were analysed in the full household contacts' cohort, and of those, 134 contacts with available longitudinal serology data and not vaccinated pre-enrolment were analysed in the serology subcohort. Household infection rate was 28·4% (95% CI 20·8-37·5) for pre-alpha-exposed contacts and 51·8% (42·5-61·0) for alpha-exposed contacts (p=0·0047). Primary cases' URT RNA viral load did not correlate with transmission, but was associated with detection of SARS-CoV-2 RNA on their hands (p=0·031). SARS-CoV-2 detected on primary cases' hands, in turn, predicted contacts' risk of infection (adjusted relative risk [aRR]=1·70 [95% CI 1·24-2·31]), as did SARS-CoV-2 RNA presence on household surfaces (aRR=1·66 [1·09-2·55]) and contacts' hands (aRR=2·06 [1·57-2·69]). In six contacts with an initial negative URT PCR result, hand-swab (n=3) and household surface-swab (n=3) PCR positivity preceded URT PCR positivity. WGS corroborated household transmission. INTERPRETATION: Presence of SARS-CoV-2 RNA on primary cases' and contacts' hands and on frequently-touched household surfaces associates with transmission, identifying these as potential vectors for spread in households. FUNDING: National Institute for Health Research Health Protection Research Unit in Respiratory Infections, Medical Research Council.

Patients with end stage kidney disease receiving in-center hemodialysis (ICHD) have had high rates of SARS-CoV-2 infection. Following infection, patients receiving ICHD frequently develop circulating antibodies to SARS-CoV-2, even with asymptomatic infection. Here, we investigated the durability and functionality of the immune responses to SARS-CoV-2 infection in patients receiving ICHD. Three hundred and fifty-six such patients were longitudinally screened for SARS-CoV-2 antibodies and underwent routine PCR-testing for symptomatic and asymptomatic infection. Patients were regularly screened for nucleocapsid protein (anti-NP) and receptor binding domain (anti-RBD) antibodies, and those who became seronegative at six months were screened for SARS-CoV-2 specific T-cell responses. One hundred and twenty-nine (36.2%) patients had detectable antibody to anti-NP at time zero, of whom 127 also had detectable anti-RBD. Significantly, at six months, 71/111 (64.0%) and 99/116 (85.3%) remained anti-NP and anti-RBD seropositive, respectively. For patients who retained antibody, both anti-NP and anti-RBD levels were reduced significantly after six months. Eleven patients who were anti-NP seropositive at time zero, had no detectable antibody at six months; of whom eight were found to have SARS-CoV-2 antigen specific T cell responses. Independent of antibody status at six months, patients with baseline positive SARS-CoV-2 serology were significantly less likely to have PCR confirmed infection over the following six months. Thus, patients receiving ICHD mount durable immune responses six months post SARS-CoV-2 infection, with fewer than 3% of patients showing no evidence of humoral or cellular immunity., Graphical abstract

BackgroundChlamydial infection is associated with tubal factor infertility (TFI); however, assessment of prior chlamydial infection and TFI is imperfect. We previously evaluated a combination of serological assays for association with TFI. We now describe the chlamydial contribution to TFI using a newer Chlamydia trachomatis Pgp3 enhanced serological (Pgp3) assay.MethodsIn our case-control study of women 19–42 years old with hysterosalpingogram-diagnosed TFI (cases) and non-TFI (controls) in two U.S. infertility clinics, we assessed possible associations and effect modifiers between Pgp3 seropositivity and TFI using adjusted odds ratios (aOR) with 95% confidence intervals (CI) stratified by race. We then estimated the adjusted chlamydia population attributable fraction (aPAF) with 95% CI of TFI.ResultsAll black (n=107) and 618 of 620 non-black women had Pgp3 results. Pgp3 seropositivity was 25.9% (19.3–33.8%) for non-black cases, 15.2% (12.3–18.7%) for non-black controls, 66.0% (95% CI 51.7–77.8%) for black cases, and 71.7% (59.2–81.5%) for black controls. Among 476 non-black women without endometriosis (n=476), Pgp3 was associated with TFI (aOR 2.6 [1.5– 4.4]), adjusting for clinic, age, and income; chlamydia TFI aPAF was 19.8% (95% CI 7.7–32.2%) in these women. Pgp3 positivity was not associated with TFI among non-black women with endometriosis nor among black women (regardless of endometriosis).ConclusionsAmong non-black infertile women without endometriosis in these clinics, 20% of TFI was attributed to chlamydia. Better biomarkers are needed to estimate chlamydia TFI PAF, especially in black women.

It is currently unknown how post-COVID-19 syndrome (PCS) may affect those infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This longitudinal study includes healthcare staff who tested positive for SARS-CoV-2 between March and April 2020, with follow-up of their antibody titers and symptoms. More than half (21 of 38) had PCS after 7–8 months. There was no statistically significant difference between initial reverse-transcription polymerase chain reaction titers or serial antibody levels between those who did and those who did not develop PCS. This study highlights the relative commonality of PCS in healthcare workers and this should be considered in vaccination scheduling and workforce planning to allow adequate frontline staffing numbers.

Associations between markers of liver and renal dysfunction and nucleotide reverse transcriptase inhibitor plasma exposure are ill-defined. As part of a large cohort study (Pharmacokinetic and Clinical Observations in People over Fifty), we analysed associations between alanine aminotransferase and estimated glomerular filtration rate results in people living with HIV on tenofovir disoproxil fumarate, emtricitabine, abacavir and lamivudine. While we found no associations between nucleotide reverse transcriptase inhibitor concentrations and alanine aminotransferase, lower estimated glomerular filtration rate values were associated with greater tenofovir, emtricitabine and lamivudine exposure, whereas abacavir showed no associations.

ObjectivesDolutegravir has replaced efavirenz as first-line treatment in universal HIV guidelines. We sought to ascertain the contributory effect of SNPs in four key genes linked to dolutegravir disposition (UGT1A1, ABCG2, CYP3A and NR1I2) on plasma dolutegravir pharmacokinetics.MethodsPaired pharmacogenetic/pharmacokinetic data from 93 subjects were analysed for association using multivariate linear regression.ResultsCo-occurring UGT1*28 and NR1I2 c.63396C>T homozygosity was associated with a 79% increase in AUC0–24 (P = 0.001; 27% if analysed individually), whilst combined ABCG2 c.421C>A and NR1I2 c.63396C>T variants were associated with a 43% increase in Cmax (P = 0.002) and a 39% increase in AUC0–24 (P = 0.002). When analysed individually, homozygosity for the NR1I2 c.63396C>T variant alleles was associated with a 28% increase in Cmax (P = 0.033) and homozygosity for the ABCG2 c.421C>A variant alleles was associated with a 28% increase in Cmax (P = 0.047). The UGT1A1*28 (rs8175347) poor metabolizer status (*28/*28; *28/*37; *37/*37) was individually associated with a 27% increase in AUC0–24 (P = 0.020). The combination of UGT1A1*28 poor metabolizer and UGT1A1*6 intermediate metabolizer statuses correlated with a 43% increase in AUC0–24 (P = 0.023).ConclusionsThis study showed a pharmacogenetic association between dolutegravir pharmacokinetics and variants in the ABCG2, UGT1A1 and NR1I2 genes, particularly when combined. Further research is warranted to confirm these associations in population-specific studies and to investigate their putative relationship with dolutegravir pharmacodynamics.

There has been much discussion on the general principle of "naturally-acquired” immunity arising from recovery from SARS CoV 2infection, and this recovery state contributing to the prevalence of immunity to reinfection, often referred to as Herd Immunity. This has led to the belief that hospital staff previously infected are safe and do not require vaccine. We demonstrate here that this may be a misplaced notion because the avidity with which the antibody from a person recovered from corona virus infection, even if of a high titre, is low. Effete antibody of low avidity is, therefore, unlikely to impact on the incidence of infection in the population, or indeed protect the individual. By contrast, a single immunisation of a previously-recovered individual by vaccine significantly increases the antibody avidity.

Introduction Immunoassays targeting different SARS-CoV-2-specific antibodies are employed for seroprevalence studies. The degree of variability between immunoassays targeting anti-nucleocapsid (anti-NP; the majority) vs the potentially neutralising anti-spike antibodies (including anti-receptor-binding domain; anti-RBD), particularly in mild or asymptomatic disease, remains unclear. Aims We aimed to explore variability in anti-NP and anti-RBD antibody detectability following mild symptomatic or asymptomatic SARS-CoV-2 infection and analyse antibody response for correlation with symptomatology. Methods A multicentre prospective cross-sectional study was undertaken (April–July 2020). Paired serum samples were tested for anti-NP and anti-RBD IgG antibodies and reactivity expressed as binding ratios (BR). Multivariate linear regression was performed analysing age, sex, time since onset, symptomatology, anti-NP and anti-RBD antibody BR. Results We included 906 adults. Antibody results (793/906; 87.5%; 95% confidence interval: 85.2–89.6) and BR strongly correlated (ρ = 0.75). PCR-confirmed cases were more frequently identified by anti-RBD (129/130) than anti-NP (123/130). Anti-RBD testing identified 83 of 325 (25.5%) cases otherwise reported as negative for anti-NP. Anti-NP presence (+1.75/unit increase; p Conclusion SARS-CoV-2 anti-RBD IgG showed significant correlation with anti-NP IgG for absolute seroconversion and BR. Higher BR were seen in symptomatic individuals, particularly those with fever. Inter-assay variability (12.5%) was evident and raises considerations for optimising seroprevalence testing strategies/studies.

BackgroundQuick, cheap and accurate point-of-care testing is urgently needed to enable frequent, large-scale testing to contain COVID-19. Lateral flow tests for antigen and antibody detection are an obvious candidate for use in community-wide testing, because they are quick and cheap relative to lab-processed tests. However, their low accuracy has limited their adoption. We develop a new methodology to increase the diagnostic accuracy of a combination of cheap, quick and inaccurate index tests with correlated or discordant outcomes, and illustrate its performance on commercially available lateral flow immunoassays (LFIAs) for Sars-CoV-2 antibody detection.Methods and FindingsWe analyze laboratory test outcomes of 300 serum samples from health care workers detected with PCR-confirmed SARS-Cov-2 infection at least 21 days prior to sample collection, and 500 pre-pandemic serum samples, from a national seroprevalence survey, tested using eight LFIAs (Abbott, Biosure/Mologic, Orientgene-Menarini, Fortress, Biopanda I, Biopanda II, SureScreen and Wondfo) and Hybrid DABA as reference test. For each of 14 two-test combinations (e.g., Abbott, Fortress) and 16 three-test combinations (e.g., Abbott, Fortress, Biosure/Mologic) used on at least 100 positive and 100 negative samples, we classify an outcome sequence – e.g., (+,–) for (Abbott, Fortress) – as positive if its combination positive predictive value (CPPV) exceeds a given threshold, set between 0 and 1. Our main outcome measures are the sensitivity and specificity of different classification rules for classifying the outcomes of a combination test. We define testing possibility frontiers which represent sensitivity and false positive rates for different thresholds. The envelope of frontiers further enables test selection.The eight index tests individually meet neither the UK Medicines and Healthcare Products Regulatory Agency’s 98% sensitivity and 98% specificity criterion, nor the US Center for Disease Control’s 99.5% specificity criterion. Among these eight tests, the highest single-test LFIA specificity is 99.4% (with a sensitivity of 65.2%) and the highest single-test LFIA sensitivity is 93.4% (with a specificity of 97.4%). Using our methodology, a two-test combination meets the UK Medicines and Healthcare Products Regulatory Agency’s criterion, achieving sensitivity of 98.4% and specificity of 98.0%. While two-test combinations meeting the US Center for Disease Control’s 99.5% specificity criterion have sensitivity below 83.6%, a three-test combination delivers a specificity of 99.6% and a sensitivity of 95.8%.ConclusionsCurrent CDC guidelines suggest combining tests, noting that “performance of orthogonal testing algorithms has not been systematically evaluated” and highlighting discordant outcomes. Our methodology combines available LFIAs to meet desired accuracy criteria, by identifying testing possibility frontiers which encompass benchmarks, enabling cost savings. Our methodology applies equally to antigen testing and can greatly expand testing capacity through combining less accurate tests, especially for use cases needing quick, accurate tests, e.g., entry to public spaces such as airports, nursing homes or hospitals.

The highly transmissible Delta variant of SARS-CoV-2 (B.1.617.2), first identified in India, is currently replacing pre-existing variants in Europe, the USA, and many other parts of the world. It is essential to monitor efficiently its spread to help guide public health policies. Genome sequencing is the gold standard for identification of Delta, but is time-consuming, expensive, and unavailable in many regions. We describe here a rapid and relatively inexpensive alternative to sequencing for specific identification of the Delta variant, by application of double-mismatch allele-specific RT-PCR (DMAS-RT-PCR). The technique exploits forward and reverse allele-specific primers, targeting two spike gene mutations, L452R and T478K, within the same amplicon. The discriminatory power of each primer is enhanced by the presence of an additional mismatch located at the fourth nucleotide from the 3′ end. Amplicons are detected in real-time by means of a conventional fluorescently-labelled hydrolysis probe. Specificity was assessed by testing a range of well characterised cell culture-derived viral isolates and clinical samples, most of which had previously been fully sequenced. In all cases the results of viral genotyping by DMAS-RT-PCR were entirely concordant with the results of sequencing, and the assay was shown to discriminate reliably between the Delta variant and other variants of concern (Alpha, B.1.1.7 and Beta, B.1.351), and ‘wild-type’ SARS-CoV-2. Other respiratory viruses, including influenza A and respiratory syncytial virus, were non-reactive in the assay. The sensitivity of DMAS-RT-PCR matched that of the diagnostic SARS-CoV-2 RT-qPCR screening assay, which targets the E gene. Several samples that could not be sequenced due to insufficient virus could successfully be genotyped by DMAS-RT-PCR. The method we describe would be simple to establish in any laboratory that has the ability to conduct PCR assays and should greatly facilitate monitoring of the spread of the Delta variant throughout the world, and its proportional representation in any SARS-CoV-2-infected population.

Objective To evaluate the performance of new lateral flow immunoassays (LFIAs) suitable for use in a national coronavirus disease 2019 (covid-19) seroprevalence programme (real time assessment of community transmission 2—React 2). Design Diagnostic accuracy study. Setting Laboratory analyses were performed in the United Kingdom at Imperial College, London and university facilities in London. Research clinics for finger prick sampling were run in two affiliated NHS trusts. Participants Sensitivity analyses were performed on sera stored from 320 previous participants in the React 2 programme with confirmed previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Specificity analyses were performed on 1000 prepandemic serum samples. 100 new participants with confirmed previous SARS-CoV-2 infection attended study clinics for finger prick testing. Interventions Laboratory sensitivity and specificity analyses were performed for seven LFIAs on a minimum of 200 serum samples from participants with confirmed SARS-CoV-2 infection and 500 prepandemic serum samples, respectively. Three LFIAs were found to have a laboratory sensitivity superior to the finger prick sensitivity of the LFIA currently used in React 2 seroprevalence studies (84%). These LFIAs were then further evaluated through finger prick testing on participants with confirmed previous SARS-CoV-2 infection: two LFIAs (Surescreen, Panbio) were evaluated in clinics in June-July 2020 and the third LFIA (AbC-19) in September 2020. A spike protein enzyme linked immunoassay and hybrid double antigen binding assay were used as laboratory reference standards. Main outcome measures The accuracy of LFIAs in detecting immunoglobulin G (IgG) antibodies to SARS-CoV-2 compared with two reference standards. Results The sensitivity and specificity of seven new LFIAs that were analysed using sera varied from 69% to 100%, and from 98.6% to 100%, respectively (compared with the two reference standards). Sensitivity on finger prick testing was 77% (95% confidence interval 61.4% to 88.2%) for Panbio, 86% (72.7% to 94.8%) for Surescreen, and 69% (53.8% to 81.3%) for AbC-19 compared with the reference standards. Sensitivity for sera from matched clinical samples performed on AbC-19 was significantly higher with serum than finger prick at 92% (80.0% to 97.7%, P=0.01). Antibody titres varied considerably among cohorts. The numbers of positive samples identified by finger prick in the lowest antibody titre quarter varied among LFIAs. Conclusions One new LFIA was identified with clinical performance suitable for potential inclusion in seroprevalence studies. However, none of the LFIAs tested had clearly superior performance to the LFIA currently used in React 2 seroprevalence surveys, and none showed sufficient sensitivity and specificity to be considered for routine clinical use.

SARS-CoV-2 spike N-terminal domain harbors a potent epitope that can be modulated by binding of natural linear tetrapyrroles., The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of heme metabolism, with nanomolar affinity. Using cryo–electron microscopy and x-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through recruitment of a metabolite.

Objectives Critical care workers were considered to be at high risk of severe acute respiratory syndrome coronavirus-2 infection from patients during the first wave of the pandemic. Staff symptoms, previous swab testing, and antibody prevalence were correlated with patient admissions to investigate this assumption. Design Cross-sectional study. Setting A large critical care department in a tertiary-care teaching hospital in London, United Kingdom. Subjects Staff working in critical care. Interventions None. Measurements and main results Participants completed a questionnaire and provided a serum sample for severe acute respiratory syndrome coronavirus-2 antibody testing over a 3-day period in April 2020. We compared the timing of symptoms in staff to the coronavirus disease 2019 patient admissions to critical care. We also identified factors associated with antibody detection. Of 625 staff, 384 (61.4%) reported previous symptoms and 124 (19.8%) had sent a swab for testing. Severe acute respiratory syndrome coronavirus-2 infection had been confirmed in 37 of those swabbed (29.8%). Overall, 21% (131/625) had detectable severe acute respiratory syndrome coronavirus-2 antibody, of whom 9.9% (13/131) had been asymptomatic. The peak onset of symptoms among staff occurred 2 weeks before the peak in coronavirus disease 2019 patient admissions. Staff who worked in multiple departments across the hospital were more likely to be seropositive. Staff with a symptomatic household contact were also more likely to be seropositive at 31.3%, compared with 16.2% in those without (p Conclusions Staff who developed coronavirus disease 2019 were less likely to have caught it from their patients in critical care. Other staff, other areas of the hospital, and the wider community are more likely sources of infection. These findings indicate that personal protective equipment was effective at preventing transmission from patients. However, staff also need to maintain protective measures away from the bedside.

BackgroundPatients with end stage kidney disease (ESKD) receiving in-centre haemodialysis (ICHD) have had high rates of SARS-CoV-2 infection. Following infection, ICHD patients frequently develop serological evidence of infection, even with asymptomatic disease. The aim of this study is to investigate the durability and functionality of immune responses to SARS-CoV-2 infection in ICHD patients.MethodsThree hundred and fifty-six ICHD patients were longitudinally screened for SARS-CoV-2 antibodies and underwent routine PCR-testing for symptomatic and asymptomatic infection. Patients were screened for nucleocapsid protein (anti-NP) and receptor binding domain (anti-RBD) antibodies. Patients who became seronegative at 6 months were investigated for SARS-CoV-2 specific T-cell responses.ResultsOne hundred and twenty-nine (36.2%) patients had detectable antibody to anti-NP at Time 0, of which 127 (98.4%) also had detectable anti-RBD. At 6 months, of 111 patients tested, 71(64.0%) and 97 (87.4%) remained anti-NP and anti-RBD seropositive respectively, pIndependent of antibody status at 6 months, patients with baseline positive SARS-CoV-2 serology were significantly less likely to have PCR confirmed infection over the following 6 months.ConclusionsICHD patients mount durable immune responses 6 months post SARS-CoV-2 infection, with SIGNIFICANCE STATEMENTFollowing infection with SARS-CoV-2, patients with end stage kidney disease (ESKD) frequently develop serological evidence of infection, even with asymptomatic disease. Patients with ESKD receiving in-centre haemodialysis (ICHD) have had high rates of SARS-CoV-2 infection. What is not known is how durable the serological responses in ESKD patients are or whether evidence of prior immune responses protect patients from reinfection. In this study of 356 ICHD patients, at 6 months following the detection of SARS-CoV-2 antibodies, fewer than 3% of patients lacked evidence of either humoral or cellular immunity. Furthermore, patients with serological evidence of infection had a significantly lower risk of being diagnosed with subsequent infection or ‘reinfection’, suggesting functional immune protection.

Objectives The wide spectrum of clinical outcomes to SARSCoV-2 exposure suggests that early immune responses play a pivotal role 1 We aim to describe early, longitudinal, local (nasal mucosal lining fluid) and systemic (peripheral blood) cytokine and cellular immune responses to SARS-CoV-2 in a symptomatic index case and their household contacts with detailed clinical and virological phenotyping We hypothesise that immune responses at symptom onset would correlate with outcomes Methods Participants from the London area are referred to INSTINCT study by general practitioners as suspected, or Public Health England as laboratory-confirmed, cases (ethical review details: IRAS 282820, approved 24 04 2020) Households are visited the day after identification and again on days 7, 15 and 28 Clinical and exposure questionnaires, samples of environment (surface swabs and air);oropharynx (swabs);nasal mucosa (synthetic absorptive matrix) and blood, and daily symptom diaries are collected Samples are analysed by PCR, serology, 20-plex cytokine assay and flow cytometry in institutional laboratories Results The index case was the first SARS-CoV-2 PCR-positive recruit of INSTINCT, confirmed on oropharyngeal swab 5 days after symptom onset Contacts 1 and 2, the spouse and daughter, became symptomatic 2 days after the index case and were confirmed PCR-positive 3 days after symptom onset The three PCR-positive individuals seroconverted during follow-up Contact 3, the son, remained asymptomatic, PCRand serology-negative throughout (figure 1a-b) None required hospitalisation Swabs of the kettle and fridge handles were positive for virus, while other household surfaces and air samples were negative Induction, peak and decline of interferonl-1 and IP-10 levels were captured in nasal mucosa, with lower serum levels (figures 1c-f) Conclusion These data demonstrate the ability of the INSTINCT household contact study to capture early immune responses in mild SARS-CoV-2 infection, not captured by COVID-19 hospital cohort studies Early nasal mucosal cytokine responses to SARS-CoV-2 infection are not reflected in serum The correlations observed provide cogent hypotheses that will be tested in the larger INSTINCT cohort, with implications for COVID-19 risk stratification, therapeutics, prophylaxis and vaccinology

There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charite assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control. Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4 % of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results. We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples. The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that the virus co-opts the haem metabolite for the evasion of humoral immunity via allosteric shielding of a sensitive epitope and demonstrate the remarkable structural plasticity of the NTD.

At the start of the UK coronavirus disease 2019 epidemic, this rare point prevalence study revealed that one-third of patients (15 of 45) in a London inpatient rehabilitation unit were found to be infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but asymptomatic. We report on 8 patients in detail, including their clinical stability, the evolution of their nasopharyngeal viral reverse-transcription polymerase chain reaction (RT-PCR) burden, and their antibody levels over time, revealing the infection dynamics by RT-PCR and serology during the acute phase. Notably, a novel serological test for antibodies against the receptor binding domain of SARS-CoV-2 showed that 100% of our asymptomatic cohort remained seropositive 3—6 weeks after diagnosis.

BackgroundAccurate antibody tests are essential to monitor the SARS-CoV-2 pandemic. Lateral flow immunoassays (LFIAs) can deliver testing at scale. However, reported performance varies, and sensitivity analyses have generally been conducted on serum from hospitalised patients. For use in community testing, evaluation of finger-prick self-tests, in non-hospitalised individuals, is required.MethodsSensitivity analysis was conducted on 276 non-hospitalised participants. All had tested positive for SARS-CoV-2 by reverse transcription PCR and were ≥21 days from symptom onset. In phase I, we evaluated five LFIAs in clinic (with finger prick) and laboratory (with blood and sera) in comparison to (1) PCR-confirmed infection and (2) presence of SARS-CoV-2 antibodies on two ‘in-house’ ELISAs. Specificity analysis was performed on 500 prepandemic sera. In phase II, six additional LFIAs were assessed with serum.Findings95% (95% CI 92.2% to 97.3%) of the infected cohort had detectable antibodies on at least one ELISA. LFIA sensitivity was variable, but significantly inferior to ELISA in 8 out of 11 assessed. Of LFIAs assessed in both clinic and laboratory, finger-prick self-test sensitivity varied from 21% to 92% versus PCR-confirmed cases and from 22% to 96% versus composite ELISA positives. Concordance between finger-prick and serum testing was at best moderate (kappa 0.56) and, at worst, slight (kappa 0.13). All LFIAs had high specificity (97.2%–99.8%).InterpretationLFIA sensitivity and sample concordance is variable, highlighting the importance of evaluations in setting of intended use. This rigorous approach to LFIA evaluation identified a test with high specificity (98.6% (95%CI 97.1% to 99.4%)), moderate sensitivity (84.4% with finger prick (95% CI 70.5% to 93.5%)) and moderate concordance, suitable for seroprevalence surveys.

BackgroundUnderstanding of the true asymptomatic rate of infection of SARS-CoV-2 is currently limited, as is understanding of the population-based seroprevalence after the first wave of COVID-19 within the UK. The majority of data thus far come from hospitalised patients, with little focus on general population cases, or their symptoms.MethodsWe undertook enzyme linked immunosorbent assay characterisation of IgM and IgG responses against SARS-CoV-2 spike glycoprotein and nucleocapsid protein of 431 unselected general-population participants of the TwinsUK cohort from South-East England, aged 19-86 (median age 48; 85% female). 382 participants completed prospective logging of 14 COVID-19 related symptoms via the COVID Symptom Study App, allowing consideration of serology alongside individual symptoms, and a predictive algorithm for estimated COVID-19 previously modelled on PCR positive individuals from a dataset of over 2 million.FindingsWe demonstrated a seroprevalence of 12% (51participants of 431). Of 48 seropositive individuals with full symptom data, nine (19%) were fully asymptomatic, and 16 (27%) were asymptomatic for core COVID-19 symptoms: fever, cough or anosmia. Specificity of anosmia for seropositivity was 95%, compared to 88% for fever cough and anosmia combined. 34 individuals in the cohort were predicted to be Covid-19 positive using the App algorithm, and of those, 18 (52%) were seropositive.InterpretationSeroprevalence amongst adults from London and South-East England was 12%, and 19% of seropositive individuals with prospective symptom logging were fully asymptomatic throughout the study. Anosmia demonstrated the highest symptom specificity for SARS-CoV-2 antibody response.FundingNIHR BRC, CDRF, ZOE global LTD, RST-UKRI/MRC

To understand the pathogenesis of low level viraemia (LLV) in HIV-infected patients on boosted protease inhibitors (PI/b), we enrolled 34 subjects with a median HIV-RNA 79 copies/mL and followed them for 15 months. Samples for next generation sequencing were collected at three time-points. Two showed resistance-associated mutations (RAMs) in the protease gene, while 95-100% had RAMs in the gag gene, which evolved in approximately a quarter of subjects, suggesting a potential clinical role of these kind of mutations.

Background : The highly transmissible Delta variant of SARS-CoV-2 (B.1.617.2), first identified in India, is currently replacing pre-existing variants in many parts of the world. To help guide public health policies it is important to monitor efficiently its spread. Genome sequencing is the gold standard for identification of Delta, but is time-consuming, expensive, and unavailable in many regions. Objective : To develop and evaluate a rapid, simple and inexpensive alternative to sequencing for Delta identification. Methods : A double-mismatch allele-specific RT-PCR (DMAS-RT-PCR) was developed. The technique exploits allele-specific primers, targeting two spike gene mutations, L452R and T478K, within the same amplicon. The discriminatory power of each primer was enhanced by an additional mismatch located at the fourth nucleotide from the 3′ end. Specificity was assessed by testing well characterised cell culture-derived viral isolates and clinical samples, most of which had previously been fully sequenced. Results : In all cases the results of viral genotyping by DMAS-RT-PCR were entirely concordant with the results of sequencing, and the assay was shown to discriminate reliably between the Delta variant and other variants (Alpha and Beta), and ‘wild-type’ SARS-CoV-2. Influenza A and RSV were non-reactive in the assay. The sensitivity of DMAS-RT-PCR matched that of the diagnostic SARS-CoV-2 RT-qPCR screening assay. Several samples that could not be sequenced due to insufficient virus were successfully genotyped by DMAS-RT-PCR. Conclusion : The method we describe would be simple to establish in any laboratory that can conduct PCR assays and should greatly facilitate monitoring of the spread of the Delta variant globally.

Background: The new SARS-CoV-2-induced disease (COVID-19) pandemic has represented a huge challenge for the health systems and has been responsible for almost 700.000 deaths worldwide as of 1st August 2020. Older age, male sex, comorbidities, ethnicity and socioeconomic factorsare risk factors for severe COVID-19. While the direct effect of solid cancer on the COVID-19 outcome has been investigated and is still debated, limited information is available on the impact of an underlying hematological malignancy on the risk of contracting SARS-CoV-2, the clinical presentation and the outcome of the infection. We report on the prevalence of SARS-CoV-2 infection in a large cohort of patients (pts) with chronic myeloid leukemia (CML). Methods: We retrospectively investigated the exposure to SARS-CoV-2 through serological testing in a single centre cohort of CML pts. Of more than 600 pts on active follow-up, we have screened 161 pts between 1st June and 27th July 2020. At each consultation, we recorded any exposure to or symptoms of SARS-CoV-2 infection in the preceding 6 months. The Imperial Hybrid DABA, a two-step double antigen binding assay (DABA) for the detection and measurement of total antibody directed to the receptor binding domain (RBD) of SARS-CoV-2 was used for analysis. The cut-off (CO) for positivity is established by adding 0.1 to the average of optical density (OD) obtained for the negative controls. A sample-OD/cut-off (S/CO) value ≥ 1 is considered reactive. Results: 161 CML pts in chronic phase underwent serological testing (median age 54 years (18-92), male n=94). Twenty pts were off TKI (post-alloSCT, n=12; in treatment free remission n=6; pregnancy=1 and intolerance n=1) and 141 were on active treatment (imatinib [n=41], dasatinib [n=38], nilotinib [n=23], bosutinib [n=21], asciminib [n=11], ponatinib [n=6] and K0706 [n=1]). The ethnic distribution was White (n=119), Asian-Indian (n= 24), Asian-Chinese (n=3), Black (n=9), Arab (n=5), Mixed Asian-White (n=1). Eighteen pts (11.2%) have tested positive (Table 1). Thirty-eight pts (23.6%) reported symptoms compatible with COVID-19 of whom 12 (31.6%) were DABA positive. Six of 123 (4.9%) asymptomatic pts also tested positive. The time from onset of symptoms to the date of testing were similar in both seropositive (105 days, range 56-180) and seronegative pts (100 days, range 21-205). The median S/CO ratio was 9.9 (1-23.3) in the symptomatic and 1.5 (1.2-21.4) in the asymptomatic pts. Among the 18 positive pts, the median age at symptom presentation was 54 years (38-75) and 12 were males. The median time from CML diagnosis was 7.6 years (0.2-20) and two pts were post-alloSCT. Ethnicity was White, Asian-Indian, Black in 8 (44.4%), 6 (33%) and 4 (22.2%), respectively. In 15 pts (83.3%) symptoms were absent or mild, moderate in 2 and severe in one patient 2 years post-alloSCT on continuing immunosuppression for GvHD. Only 2/18 had PCR tests for SARS-CoV-2 at the time of infection and both were positive. Of the 16 pts not tested for antigen, 2 had been in close contact with a family member with a PCR-confirmed SARS-CoV-2 infection. In the mild cases, the most frequent symptoms were fever (n=7) and cough (n=6), followed by fatigue (n=2), myalgia (n=2) and anosmia (n=2). One patient presented only with acute limb ischemia, derived from thrombus in the superficial femoral artery. One patient had radiological features of COVID-19 pneumonia, but did not require hospitalization. The only patient with severe COVID-19 developed pneumonia requiring CPAP, was refractory to tocilizumab (anti-IL6), but recovered after starting ruxolitinib (JAK2-inhibitor). The median time to complete resolution of symptoms was 21 days (7-56). Conclusions: The prevalence of infection in our CML pts is similar to that of the overall population, which suggests that they are capable of mounting appropriate antibody response against SARS-CoV-2. Importantly, in seropositive pts symptoms were mild. Of note is the higher prevalence in the BAME community and underlines the potential role of socioeconomic factors in disease transmission. Expansion of this CML cohort and serial antibody testing in seropositive patients will provide further information regarding prevalence and durability of serological responses. Disclosures Milojkovic: Incyte: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Apperley:Bristol Myers Squibb: Honoraria, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Fever is the most common reason that children present to Emergency Departments in the UK. Clinical signs and symptoms suggestive of bacterial infection are often non-specific, and there is no definitive test for the accurate diagnosis of infection. As a result, many children are prescribed antibiotics often unnecessarily, while others with life-threatening bacterial infections can remain untreated. The ‘omics’ approaches to identifying biomarkers from the host-response to bacterial infection are promising. In this study, lipidomic analysis was carried out with plasma samples obtained from febrile children with confirmed bacterial infection (n=20) and confirmed viral infection (n=20). We show for the first time that bacterial and viral infection elicit distinct changes in the host lipidome. Glycerophosphoinositol, sphingomyelin, lysophosphotidylcholine and cholesterol sulfate were increased in the confirmed virus infected group, while fatty acids, glycerophosphocholine, glycerophosphoserine, lactosylceramide and bilirubin were increased in cases with confirmed bacterial infection. A combination of three lipids achieved the area under the receiver operating characteristic (ROC) curve of 0.918 (95% CI 0.835 to 1). This pilot study demonstrates the potential of metabolic biomarkers to assist clinicians in distinguishing bacterial from viral infection in febrile children, to facilitate effective clinical management and to the limit inappropriate use of antibiotics.

IntroductionSerological testing can augment delayed case identification programmes for Severe Acute Respiratory Syndrome coronoravirus-2 (SARS-CoV-2). Immunoassays employ anti-nucleocapsid (anti-NP; the majority) or potentially neutralising anti-spike (including anti-receptor binding domain; anti-RBD) antibody targets, yet correlation between assays and variability arising from disease symptomatology remains unclear. We explore these possibly differential immune responses across the disease spectrum.MethodsA multicentre prospective study was undertaken via a SARS-CoV-2 delayed case identification programme (08 May-11 July 2020). Matched samples were tested for anti-NP and anti-RBD (utilising an ‘inhouse’ double-antigen bridged assay), reactivity expressed as test/cut-off binding ratios (BR) and results compared. A multivariate linear regression model analysed age, sex, symptomatology, PCR positivity, anti-NP, and anti-RBD BRs. Participants were followed up for possible reinfection.Results902 individuals underwent matched testing; 109 were SARS-CoV-2 PCR swab positive. Anti-NP, anti-RBD immunoassay agreement was 87.5% (95% CI 85.3–89.6), with BRs strongly correlated (R=0.75). PCR confirmed cases were more frequently identified by anti-RBD (sensitivity 108/109, 99.1%, 95% CI 95.0–100.0) than anti-NP (102/109, 93.6%, 95% CI 87.2–97.4). Anti-RBD identified an additional 83/325 (25.5%) cases in those seronegative for anti-NP. Presence of anti-NP (pConclusionsSARS-CoV-2 anti-RBD shows significant correlation with anti-NP for absolute seroconversion, and BRs. Higher BRs are seen in symptomatic individuals with significantly higher levels seen in those with fever and anosmia. The degree of discordant results (12.5%) limits the use of anti-NP as a stand-alone for delayed case finding programmes. Similarly, this discordance limits the utility of non-neutralising anti-NP assays in place of potentially neutralising anti-RBD to infer possible immunity.** this abstract presentation was awarded an Honourable Mention

Sequences necessary for transduction of human endogenous retrovirus (HERV)-K con , a consensus of the HERV-K(HML-2) family, were analyzed and found to reside in the leader/ gag region. They act in an orientation-dependent way and consist of at least two sites working together. Having defined these sequences, we exploited this information to produce a simple system to investigate to what extent virions of HERV-K con , murine leukemia virus, and HIV-1 have the ability to transduce each other's genomes, leading to potential contamination of gene therapy vectors.

BackgroundSeroprevalence surveys of Chlamydia trachomatis (CT) antibodies are promising for estimating age-specific CT cumulative incidence, however accurate estimates require improved understanding of antibody response to CT infection.MethodsWe used GUMCAD, England's national sexually transmitted infection (STI) surveillance system, to select sera taken from female STI clinic attendees on the day of or after a chlamydia diagnosis. Serum specimens were collected from laboratories and tested anonymously on an indirect and a double-antigen ELISA, both of which are based on the CT-specific Pgp3 antigen. We used cross-sectional and longitudinal descriptive analyses to explore the relationship between seropositivity and a) cumulative number of chlamydia diagnoses and b) time since most recent chlamydia diagnosis.Results919 samples were obtained from visits when chlamydia was diagnosed and 812 during subsequent follow-up visits. Pgp3 seropositivity using the indirect ELISA increased from 57.1% (95% confidence interval: 53.2-60.7) on the day of a first-recorded chlamydia diagnosis to 89.6% (95%CI: 79.3-95.0) on the day of a third or higher documented diagnosis. With the double-antigen ELISA, the increase was from 61.1% (95%CI: 53.2-60.7) to 97.0% (95%CI: 88.5-99.3). Seropositivity decreased with time since CT diagnosis on only the indirect assay, to 49.3% (95%CI: 40.9-57.7) two or more years after a first diagnosis and 51.9% (95%CI: 33.2-70.0) after a repeat diagnosis.ConclusionSeropositivity increased with cumulative number of infections, and decreased over time after diagnosis on the indirect ELISA, but not on the double-antigen ELISA. This is the first study to demonstrate the combined impact of number of chlamydia diagnoses, time since diagnosis, and specific ELISA on Pgp3 seropositivity. Our findings are being used to inform models estimating age-specific chlamydia incidence over time using serial population-representative serum sample collections, to enable accurate public health monitoring of chlamydia.

BackgroundDemographic data show an increasingly aging human immunodeficiency virus (HIV) population worldwide. Recent concerns over dolutegravir-related neuropsychiatric toxicity have emerged, particularly amongst older people living with HIV (PLWH). We describe the pharmacokinetics (PK) of dolutegravir (DTG) 50 mg once daily in PLWH aged 60 and older. Additionally, to address calls for prospective neuropsychiatric toxicodynamic data, we evaluated changes in sleep quality and cognitive functioning in this population after switching to abacavir (ABC)/lamivudine (3TC)/DTG over 6 months.MethodsPLWH ≥60 years with HIV-viral load ResultsIn total, 43 participants enrolled, and 40 completed the PK phase. Overall, 5 discontinued (2 due to sleep-related adverse events, 4.6%). DTG maximum concentration (Cmax) was significantly higher in patients ≥60 years old versus controls (geometric mean 4246 ng/mL versus 3402 ng/mL, P = .005). In those who completed day 180 (n = 38), sleep impairment (Pittsburgh Sleep Quality Index) was marginally higher at day 28 (P = .02), but not at days 90 or 180. Insomnia, daytime functioning, and fatigue test scores did not change statistically over time.ConclusionsDTG Cmax was significantly higher in older PLWH. Our data provides clinicians with key information on the safety of prescribing DTG in older PLWH.

Introduction Genital infection with Chlamydia trachomatis (CT) is the most commonly-diagnosed bacterial sexually transmitted infection in England. The National Chlamydia Screening Programme (NCSP) was implemented nationwide in 2008, offering opportunistic CT testing to people under 25. Not all chlamydia infections result in a lasting antibody response, however, monitoring age-specific seroprevalence of antibodies against CT over time may offer insights into the impact of this intervention. We explored trends in seroprevalence from 2007 up to 2015. Methods Samples were obtained from the PHE Seroepidemiology Unit, which collects unlinked, anonymous, residual sera submitted to laboratories in England for routine investigations. Samples known to come from GUM clinics were excluded. Sera from 2007–2015 from women aged 15–30 (n=9,798) were tested using an indirect IgG ELISA for chlamydia Pgp3 antibody. Women in 2007 had limited exposure to the NCSP, increasing over time. Age-standardised seroprevalence was calculated for 17–24 year-olds using 2015 population data. Samples were classified by the number of years individuals were eligible for the screening programme, based on year of birth. Results Age-standardised seroprevalence among 17–24 year-olds varied, being highest at 20.3% (95% CI 17.2–23.4) in 2007 and lowest at 15.5% (95% CI 10.0–20.9) in 2015, although no clear trend was seen. Although incomplete data were available for those with ‘limited’ and ‘high’ exposure to the NCSP, age-specific seroprevalence did not vary by exposure to NCSP. Conclusion There was no evidence that age-specific seroprevalence varied by exposure to the NSCP. Interpretation of this is complicated by the potential effects of antibody prevalence waning over time, and being affected by factors such as treatment and re-infection. Other limitations include a high number (86.2%) of specimens from ‘unknown’ source which could have been from GUM clinics. Multi-parameter evidence synthesis models are being developed to explore the use of these data to estimate incidence.

Background: Although understanding chlamydia incidence assists prevention and control, analyses based on diagnosed infections may distort the findings. Therefore, we determined incidence and examined risks in a birth cohort based on self-reports and serology.Methods: Self-reported chlamydia and behavior data were collected from a cohort born in New Zealand in 1972/3 on several occasions to age 38 years. Sera drawn at ages 26, 32, and 38 years were tested for antibodies to Chlamydia trachomatis Pgp3 antigen using a recently developed assay, more sensitive in women (82.9%) than men (54.4%). Chlamydia incidence by age period (first coitus to age 26, 26-32, and 32-38 years) was calculated combining self-reports and serostatus and risk factors investigated by Poisson regression.Results: By age 38 years, 32.7% of women and 20.9% of men had seroconverted or self-reported a diagnosis. The highest incidence rate was to age 26, 32.7 and 18.4 years per 1000 person-years for women and men, respectively. Incidence rates increased substantially with increasing number of sexual partners. After adjusting age period incidence rates for partner numbers, a relationship with age was not detected until 32 to 38 years, and then only for women.Conclusions: Chlamydia was common in this cohort by age 38, despite the moderate incidence rates by age period. The strongest risk factor for incident infection was the number of sexual partners. Age, up to 32 years, was not an independent factor after accounting for partner numbers, and then only for women. Behavior is more important than age when considering prevention strategies.

IntroductionIt is known that several viral species can induce parkinsonism in animals and humans, but no specific virus has been discovered in the classical human form of PD.MethodsTo investigate this further, we used next generation sequencing (NGS) to analyse post-mortem transcriptomes of substantia nigra (SN) and olfactory bulb (OB) in four PD cases and four multiple sclerosis patients who acted as positive controls. Bioinformatic analysis of the data set removed the majority of human transcripts and the remaining sequence data were compared to existing viral sequence data sets to search for signature viral sequences.ResultsDespite high read numbers and good quality NGS data, no viral or bacterial transcripts could be identified from either of the tissues examined. The few matches to existing viral databases were to viruses which rarely, if ever, infect humans and no multiple hits (matches to more than one gene of the same virus) were observed. Similarly, no bacterial sequences were found although it should be noted the analysis was carried out at the mRNA level.ConclusionThis small but detailed analysis provided no evidence of RNA viral signatures in the OB or SN in the four samples taken from Parkinson’s disease brains. It is still possible that other brain areas known to display Lewy body pathology may contain viral sequences.

[This corrects the article DOI: 10.1371/journal.pone.0208652.].

Establishment of the stable provirus is an essential step in retroviral replication, orchestrated by integrase (IN), a virus-derived enzyme. Until now, available structural information was limited to the INs of human immunodeficiency virus type 1 (HIV-1), avian sarcoma virus (ASV) and their close orthologs from the Lentivirus and Alpharetrovirus genera. Here, we characterized the in vitro activity of the prototype foamy virus (PFV) IN from the Spumavirus genus and determined the three-dimensional structure of its catalytic core domain (CCD). Recombinant PFV IN displayed robust and almost exclusively concerted integration activity in vitro utilizing donor DNA substrates as short as 16 bp, underscoring its significance as a model for detailed structural studies. Comparison of the HIV-1, ASV and PFV CCD structures highlighted both conserved as well as unique structural features such as organization of the active site and the putative host factor binding face. Despite possessing very limited sequence identity to its HIV counterpart, PFV IN was sensitive to HIV IN strand transfer inhibitors, suggesting that this class of inhibitors target the most conserved features of retroviral IN-DNA complexes.

Three human cell lines used in human immunodeficiency virus research were found to be contaminated with previously undetected retroviruses. On the bases of partial nucleotide sequence, capsid protein antigenicity, vector mobilization, and receptor usage studies, these contaminants were shown to be replication competent and to belong to theGammaretrovirusgenus. While the TZM-bl cells harbor ecotropic murine leukemia virus (MLV), Jurkat J6 cells were found to release xenotropic MLV and the A3.01/F7 cells to produce gibbon ape leukemia virus. These findings highlight the importance of routine testing of cell lines for retrovirus contamination to prevent potential experimental artifacts and allow correct biohazard assessment.