Genomic and geographical structure of human cytomegalovirus.

Human cytomegalovirus (CMV) has infected humans since the origin of our species and currently infects most of the world's population. Variability between CMV genomes is the highest of any human herpesvirus, yet large portions of the genome are conserved. Here, we show that the genome encodes 74 regions of relatively high variability each with 2 to 8 alleles. We then identified two patterns in the CMV genome. Conserved parts of the genome and a minority (32) of variable regions show geographic population structure with evidence for African or European clustering, although hybrid strains are present. We find no evidence that geographic segregation has been driven by host immune pressure affecting known antigenic sites. Forty-two variable regions show no geographical structure, with similar allele distributions across different continental populations. These "nongeographical" regions are significantly enriched for genes encoding immunomodulatory functions suggesting a core functional importance. We hypothesize that at least two CMV founder populations account for the geographical differences that are largely seen in the conserved portions of the genome, although the timing of separation and direction of spread between the two are not clear. In contrast, the similar allele frequencies among 42 variable regions of the genome, irrespective of geographical origin, are indicative of a second evolutionary process, namely balancing selection that may preserve properties critical to CMV biological function. Given that genetic differences between CMVs are postulated to alter immunogenicity and potentially function, understanding these two evolutionary processes could contribute important information for the development of globally effective vaccines and the identification of novel drug targets.

Persistent low-level variants in a subset of viral genes are highly predictive of poor outcome in immunocompromised patients with cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus is the most common and serious opportunistic infection after solid organ and haematopoietic stem cell transplantation. In this study, we used whole-genome cytomegalovirus data to investigate viral factors associated with the clinical outcome. METHODS: We sequenced cytomegalovirus samples from 16 immunocompromised paediatric patients with persistent viraemia. 8/16 patients died of complications due to cytomegalovirus infection. We also sequenced samples from 35 infected solid organ adult recipients of whom one died with cytomegalovirus infection. RESULTS: We showed that samples from both groups have fixed variants at resistance sites and mixed infections. NGS sequencing also revealed non-fixed variants at resistance sites in most of the patients who died (6/9). A machine learning approach identified 10 genes with non-fixed variants in these patients. These genes formed a viral signature which discriminated patients with cytomegalovirus infection who died from those that survived with high accuracy (AUC=0.96). Lymphocyte numbers for a subset of patients showed no recovery post-transplant in the patients who died. CONCLUSIONS: We hypothesise that the viral signature identified in this study may be a useful biomarker for poor response to antiviral drug treatment and indirectly for poor T cell function, potentially identifying early, those patients requiring non-pharmacological interventions.

Emergence of Novel Norovirus GII.4 Variant.

We detected a novel GII.4 variant with an amino acid insertion at the start of epitope A in viral protein 1 of noroviruses from the United States, Gabon, South Africa, and the United Kingdom collected during 2017-2022. Early identification of GII.4 variants is crucial for assessing pandemic potential and informing vaccine development.

Favipiravir induces HuNoV viral mutagenesis and infectivity loss with clinical improvement in immunocompromised patients.

Chronic human norovirus (HuNoV) infections in immunocompromised patients result in severe disease, yet approved antivirals are lacking. RNA-dependent RNA polymerase (RdRp) inhibitors inducing viral mutagenesis display broad-spectrum in vitro antiviral activity, but clinical efficacy in HuNoV infections is anecdotal and the potential emergence of drug-resistant variants is concerning. Upon favipiravir (and nitazoxanide) treatment of four immunocompromised patients with life-threatening HuNoV infections, viral whole-genome sequencing showed accumulation of favipiravir-induced mutations which coincided with clinical improvement although treatment failed to clear HuNoV. Infection of zebrafish larvae demonstrated drug-associated loss of viral infectivity and favipiravir treatment showed efficacy despite occurrence of RdRp variants potentially causing favipiravir resistance. This indicates that within-host resistance evolution did not reverse loss of viral fitness caused by genome-wide accumulation of sequence changes. This off-label approach supports the use of mutagenic antivirals for treating prolonged RNA viral infections and further informs the debate surrounding their impact on virus evolution.

Molnupiravir plus usual care versus usual care alone as early treatment for adults with COVID-19 at increased risk of adverse outcomes (PANORAMIC): an open-label, platform-adaptive randomised controlled trial.

BACKGROUND: The safety, effectiveness, and cost-effectiveness of molnupiravir, an oral antiviral medication for SARS-CoV-2, has not been established in vaccinated patients in the community at increased risk of morbidity and mortality from COVID-19. We aimed to establish whether the addition of molnupiravir to usual care reduced hospital admissions and deaths associated with COVID-19 in this population. METHODS: PANORAMIC was a UK-based, national, multicentre, open-label, multigroup, prospective, platform adaptive randomised controlled trial. Eligible participants were aged 50 years or older-or aged 18 years or older with relevant comorbidities-and had been unwell with confirmed COVID-19 for 5 days or fewer in the community. Participants were randomly assigned (1:1) to receive 800 mg molnupiravir twice daily for 5 days plus usual care or usual care only. A secure, web-based system (Spinnaker) was used for randomisation, which was stratified by age (<50 years vs ≥50 years) and vaccination status (yes vs no). COVID-19 outcomes were tracked via a self-completed online daily diary for 28 days after randomisation. The primary outcome was all-cause hospitalisation or death within 28 days of randomisation, which was analysed using Bayesian models in all eligible participants who were randomly assigned. This trial is registered with ISRCTN, number 30448031. FINDINGS: Between Dec 8, 2021, and April 27, 2022, 26 411 participants were randomly assigned, 12 821 to molnupiravir plus usual care, 12 962 to usual care alone, and 628 to other treatment groups (which will be reported separately). 12 529 participants from the molnupiravir plus usual care group, and 12 525 from the usual care group were included in the primary analysis population. The mean age of the population was 56·6 years (SD 12·6), and 24 290 (94%) of 25 708 participants had had at least three doses of a SARS-CoV-2 vaccine. Hospitalisations or deaths were recorded in 105 (1%) of 12 529 participants in the molnupiravir plus usual care group versus 98 (1%) of 12 525 in the usual care group (adjusted odds ratio 1·06 [95% Bayesian credible interval 0·81-1·41]; probability of superiority 0·33). There was no evidence of treatment interaction between subgroups. Serious adverse events were recorded for 50 (0·4%) of 12 774 participants in the molnupiravir plus usual care group and for 45 (0·3%) of 12 934 in the usual care group. None of these events were judged to be related to molnupiravir. INTERPRETATION: Molnupiravir did not reduce the frequency of COVID-19-associated hospitalisations or death among high-risk vaccinated adults in the community. FUNDING: UK National Institute for Health and Care Research.

Varicella-Zoster Virus-Genetics, Molecular Evolution and Recombination.

This chapter first details the structure, organization and coding content of the VZV genome to provide a foundation on which the molecular evolution of the virus can be projected. We subsequently describe the evolution of molecular profiling approaches from restriction fragment length polymorphisms to single nucleotide polymorphism profiling to modern day high-throughput sequencing approaches. We describe how the application of these methodologies led to our current model of VZV phylogeograpy including the number and structure of geographic clades and the role of recombination in reshaping these.

Herpes Zoster and Risk of Incident Parkinson's Disease in US Veterans: A Matched Cohort Study.

BACKGROUND: Although some systemic infections are associated with Parkinson's disease (PD), the relationship between herpes zoster (HZ) and PD is unclear. OBJECTIVE: The objective is to investigate whether HZ is associated with incident PD risk in a matched cohort study using data from the US Department of Veterans Affairs. METHODS: We compared the risk of PD between individuals with incident HZ matched to up to five individuals without a history of HZ using Cox proportional hazards regression. In sensitivity analyses, we excluded early outcomes. RESULTS: Among 198,099 individuals with HZ and 976,660 matched individuals without HZ (median age 67.0 years (interquartile range [IQR 61.4-75.7]); 94% male; median follow-up 4.2 years [IQR 1.9-6.6]), HZ was not associated with an increased risk of incident PD overall (adjusted HR 0.95, 95% CI 0.90-1.01) or in any sensitivity analyses. CONCLUSION: We found no evidence that HZ was associated with increased risk of incident PD in this cohort. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Blood donation screening for hepatitis B virus core antibodies: The importance of confirmatory testing and initial implication for rare blood donor groups.

BACKGROUND AND OBJECTIVES: Exclusion of blood donors with hepatitis B virus (HBV) core antibodies (anti-HBc) prevents transfusion-transmitted HBV infection but can lead to significant donor loss. As isolated anti-HBc positivity does not always indicate true past HBV infection, we have investigated the effectiveness of confirmatory anti-HBc testing and the representation of rare blood groups in anti-HBc-positive donors. MATERIALS AND METHODS: Three hundred ninety-seven HBV surface antigen-negative and anti-HBc initially reactive blood donor samples were tested by five different anti-HBc assays. RESULTS: Eighty percentage of samples reactive in Architect anti-HBc assay were positive by the Murex assay and anti-HBc neutralization. Eleven out of 397 samples showed discordant results in supplementary testing from the Murex confirmatory test result, and five remained undetermined following extensive serological testing. Thirty-eight percentage of anti-HBc-positive donors identified as minority ethnic groups compared with 11% representation in anti-HBc-negative donors (p < 0.0001); the frequency of the Ro blood group in anti-HBc-positive donors was 18 times higher in non-white ethnic groups. CONCLUSION: Using two anti-HBc assays effectively enabled the identification of HBV-exposed and potentially infectious donors, their deferral and potential clinical follow-up. However, the exclusion of confirmed anti-HBc-positive donors will still impact the supply of rare blood such as Ro.

Persistent SARS-CoV-2 PCR Positivity Despite Anti-viral Treatment in Immunodeficient Patients.

PURPOSE: COVID-19 infection in immunodeficient individuals can result in chronically poor health, persistent or relapsing SARS-CoV-2 PCR positivity, and long-term infectious potential. While clinical trials have demonstrated promising outcomes using anti-SARS-CoV-2 medicines in immunocompetent hosts, their ability to achieve sustained viral clearance in immunodeficient patients remains unknown. We therefore aimed to study long-term virological outcomes in patients treated at our centre. METHODS: We followed up immunocompromised inpatients treated with casirivimab-imdevimab (Ronapreve) between September and December 2021, and immunocompromised patients who received sotrovimab, molnupiravir, nirmatrelvir/ritonavir (Paxlovid), or no treatment from December 2021 to March 2022. Nasopharyngeal swab and sputum samples were obtained either in hospital or in the community until sustained viral clearance, defined as 3 consecutive negative PCR samples, was achieved. Positive samples were sequenced and analysed for mutations of interest. RESULTS: We observed sustained viral clearance in 71 of 103 patients, none of whom died. Of the 32/103 patients where sustained clearance was not confirmed, 6 died (between 2 and 34 days from treatment). Notably, we observed 25 cases of sputum positivity despite negative nasopharyngeal swab samples, as well as recurrence of SARS-CoV-2 positivity following a negative sample in 12 cases. Patients were then divided into those who cleared within 28 days and those with PCR positivity beyond 28 days. We noted lower B cell counts in the group with persistent PCR positivity (mean (SD) 0.06 (0.10) ×109/L vs 0.22 (0.28) ×109/L, p = 0.015) as well as lower IgA (median (IQR) 0.00 (0.00-0.15) g/L vs 0.40 (0.00-0.95) g/L, p = 0.001) and IgM (median (IQR) 0.05 (0.00-0.28) g/L vs 0.35 (0.10-1.10) g/L, p = 0.005). No differences were seen in CD4+ or CD8+ T cell counts. Antiviral treatment did not impact risk of persistent PCR positivity. CONCLUSION: Persistent SARS-CoV-2 PCR positivity is common among immunodeficient individuals, especially those with antibody deficiencies, regardless of anti-viral treatment. Peripheral B cell count and serum IgA and IgM levels are predictors of viral persistence.

Ultrasensitive PCR system for HBV DNA detection: Risk stratification for occult hepatitis B virus infection in English blood donors.

Occult hepatitis B (HBV) infection (OBI), characterized by low viral loads, accounts for much of the risk of HBV transfusion-transmitted infection. With anticore antibodies (anti-HBc) screening introduced in England, the imperative to identify OBI donors has increased. We aimed to develop an ultra-sensitive PCR system and investigate risk factors for HBV DNA presence in blood donations. Seven extraction methods and three PCR assays were compared. The optimal system was sought to determine HBV DNA presence in anti-HBc-positive donations. Predictors of DNA positivity were subsequently investigated. Extraction from 5 mL of plasma increased sample representation and resulted in HBV DNA detection in low viral load samples (~0.5 IU/mL). Screening of 487 763 donations in 2022 identified two OBI donors and 2042 anti-HBc-positive donors, 412 of the latter with anti-HBs < 100 mIU/mL. Testing of 134 anti-HBc-positive donations utilizing the 5 mL extraction method identified two further HBV DNA-positive donations. Higher anti-HBc titer and anti-HBs negativity were significant predictors of DNA detectability in anti-HBc-positive donations. An ultrasensitive PCR assay identified potentially infectious donations increasing HBV DNA detection in anti-HBc-positive donors from 0.5% to 1.9%. Anti-HBc titers may further complement the risk stratification for DNA positivity in anti-HBc screening and minimize unnecessary donor deferral.

Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2-infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

Simultaneous quantitation of favipiravir and its hydroxide metabolite in human plasma and hamster matrices using a UPLC-MS/MS method.

Favipiravir, a broad-spectrum RNA-dependent RNA polymerase inhibitor, is currently being evaluated in preclinical and clinical studies for the treatment of various infectious diseases including COVID-19. We developed an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the quantification of favipiravir and its hydroxide metabolite (M1), in human and hamster biological matrices. Analytes were separated on an Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm) after a simple protein precipitation with acetonitrile. The mobile phase consisted of water and methanol, each containing 0.05% formic acid. Experiments were performed using electrospray ionization in the positive and negative ion mode, with protonated molecules used as the precursor ion and a total run time of 6 min. The MS/MS response was linear over the concentration ranges from 0.5-100 µg/ml for favipiravir and 0.25-30 µg/ml for M1. Intra- and inter-day accuracy and precision were within the recommended limits of the European Medicines Agency guidelines. No significant matrix effect was observed, and the method was successfully applied to inform favipiravir dose adjustments in six immunocompromised children with severe RNA viral infections. In conclusion, the UPLC-MS/MS assay is suitable for quantification of favipiravir over a wide range of dosing regimens, and can easily be adapted to other matrices and species.

Genomics of Ocular Chlamydia trachomatis After 5 Years of SAFE Interventions for Trachoma in Amhara, Ethiopia.

BACKGROUND: To eliminate trachoma as a public health problem, the World Health Organization recommends the SAFE (surgery, antibiotics, facial cleanliness, and environmental improvement) strategy. As part of the SAFE strategy in the Amhara Region, Ethiopia, the Trachoma Control Program distributed >124 million doses of antibiotics between 2007 and 2015. Despite this, trachoma remained hyperendemic in many districts and a considerable level of Chlamydia trachomatis (Ct) infection was evident. METHODS: We utilized residual material from Abbott m2000 Ct diagnostic tests to sequence 99 ocular Ct samples from Amhara and investigated the role of Ct genomic variation in continued transmission of Ct. RESULTS: Sequences were typical of ocular Ct at the whole-genome level and in tissue tropism-associated genes. There was no evidence of macrolide resistance in this population. Polymorphism around the ompA gene was associated with village-level trachomatous inflammation-follicular prevalence. Greater ompA diversity at the district level was associated with increased Ct infection prevalence. CONCLUSIONS: We found no evidence for Ct genomic variation contributing to continued transmission of Ct after treatment, adding to evidence that azithromycin does not drive acquisition of macrolide resistance in Ct. Increased Ct infection in areas with more ompA variants requires longitudinal investigation to understand what impact this may have on treatment success and host immunity.

Post-Vaccination Coronavirus Disease 2019: A Case-Control Study and Genomic Analysis of 119 Breakthrough Infections in Partially Vaccinated Individuals.

BACKGROUND: Post-vaccination infections challenge the control of the coronavirus disease 2019 (COVID-19) pandemic. METHODS: We matched 119 cases of post-vaccination severe acute respiratory syndrome coronavirus 2 infection with BNT162b2 mRNA or ChAdOx1 nCOV-19 to 476 unvaccinated patients with COVID-19 (September 2020-March 2021) according to age and sex. Differences in 60-day all-cause mortality, hospital admission, and hospital length of stay were evaluated. Phylogenetic, single-nucleotide polymorphism (SNP), and minority variant allele (MVA) full-genome sequencing analysis was performed. RESULTS: Overall, 116 of 119 cases developed COVID-19 post-first vaccination dose (median, 14 days). Thirteen of 119 (10.9%) cases and 158 of 476 (33.2%) controls died (P < .001), corresponding to the 4.5 number needed to treat (NNT). Multivariably, vaccination was associated with a 69.3% (95% confidence interval [CI]: 45.8 to 82.6) relative risk (RR) reduction in mortality. Similar results were seen in subgroup analysis for patients with infection onset ≥14 days after first vaccination and across vaccine subgroups. Hospital admissions (odds ratio, 0.80; 95% CI: .51 to 1.28) and length of stay (-1.89 days; 95% CI: -4.57 to 0.78) were lower for cases, while cycle threshold values were higher (30.8 vs 28.8, P = .053). B.1.1.7 was the predominant lineage in cases (100 of 108, 92.6%) and controls (341 of 446, 76.5%). Genomic analysis identified 1 post-vaccination case that harbored the E484K vaccine-escape mutation (B.1.525 lineage). CONCLUSIONS: Previous vaccination reduces mortality when B.1.1.7 is the predominant lineage. No significant lineage-specific genomic changes during phylogenetic, SNP, and MVA analysis were detected.

Long-Term Persistence of Spike Protein Antibody and Predictive Modeling of Antibody Dynamics After Infection With Severe Acute Respiratory Syndrome Coronavirus 2.

BACKGROUND: Antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been shown to neutralize the virus in vitro and prevent disease in animal challenge models on reexposure. However, the current understanding of SARS-CoV-2 humoral dynamics and longevity is conflicting. METHODS: The COVID-19 Staff Testing of Antibody Responses Study (Co-Stars) prospectively enrolled 3679 healthcare workers to comprehensively characterize the kinetics of SARS-CoV-2 spike protein (S), receptor-binding domain, and nucleoprotein (N) antibodies in parallel. Participants screening seropositive had serial monthly serological testing for a maximum of 7 months with the Meso Scale Discovery Assay. Survival analysis determined the proportion of seroreversion, while 2 hierarchical gamma models predicted the upper and lower bounds of long-term antibody trajectory. RESULTS: A total of 1163 monthly samples were provided from 349 seropositive participants. At 200 days after symptoms, >95% of participants had detectable S antibodies, compared with 75% with detectable N antibodies. S antibody was predicted to remain detectable in 95% of participants until 465 days (95% confidence interval, 370-575 days) using a "continuous-decay" model and indefinitely using a "decay-to-plateau" model to account for antibody secretion by long-lived plasma cells. S-antibody titers were correlated strongly with surrogate neutralization in vitro (R2 = 0.72). N antibodies, however, decayed rapidly with a half-life of 60 days (95% confidence interval, 52-68 days). CONCLUSIONS: The Co-Stars data presented here provide evidence for long-term persistence of neutralizing S antibodies. This has important implications for the duration of functional immunity after SARS-CoV-2 infection. In contrast, the rapid decay of N antibodies must be considered in future seroprevalence studies and public health decision-making. This is the first study to establish a mathematical framework capable of predicting long-term humoral dynamics after SARS-CoV-2 infection. CLINICAL TRIALS REGISTRATION: NCT04380896.

Evolutionary History of Endogenous Human Herpesvirus 6 Reflects Human Migration out of Africa.

Human herpesvirus 6A and 6B (HHV-6) can integrate into the germline, and as a result, ∼70 million people harbor the genome of one of these viruses in every cell of their body. Until now, it has been largely unknown if 1) these integrations are ancient, 2) if they still occur, and 3) whether circulating virus strains differ from integrated ones. Here, we used next-generation sequencing and mining of public human genome data sets to generate the largest and most diverse collection of circulating and integrated HHV-6 genomes studied to date. In genomes of geographically dispersed, only distantly related people, we identified clades of integrated viruses that originated from a single ancestral event, confirming this with fluorescent in situ hybridization to directly observe the integration locus. In contrast to HHV-6B, circulating and integrated HHV-6A sequences form distinct clades, arguing against ongoing integration of circulating HHV-6A or "reactivation" of integrated HHV-6A. Taken together, our study provides the first comprehensive picture of the evolution of HHV-6, and reveals that integration of heritable HHV-6 has occurred since the time of, if not before, human migrations out of Africa.

Favipiravir, lopinavir-ritonavir, or combination therapy (FLARE): A randomised, double-blind, 2 × 2 factorial placebo-controlled trial of early antiviral therapy in COVID-19.

BACKGROUND: Early antiviral treatment is effective for Coronavirus Disease 2019 (COVID-19) but currently available agents are expensive. Favipiravir is routinely used in many countries, but efficacy is unproven. Antiviral combinations have not been systematically studied. We aimed to evaluate the effect of favipiravir, lopinavir-ritonavir or the combination of both agents on Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) viral load trajectory when administered early. METHODS AND FINDINGS: We conducted a Phase 2, proof of principle, randomised, placebo-controlled, 2 × 2 factorial, double-blind trial of ambulatory outpatients with early COVID-19 (within 7 days of symptom onset) at 2 sites in the United Kingdom. Participants were randomised using a centralised online process to receive: favipiravir (1,800 mg twice daily on Day 1 followed by 400 mg 4 times daily on Days 2 to 7) plus lopinavir-ritonavir (400 mg/100 mg twice daily on Day 1, followed by 200 mg/50 mg 4 times daily on Days 2 to 7), favipiravir plus lopinavir-ritonavir placebo, lopinavir-ritonavir plus favipiravir placebo, or both placebos. The primary outcome was SARS-CoV-2 viral load at Day 5, accounting for baseline viral load. Between 6 October 2020 and 4 November 2021, we recruited 240 participants. For the favipiravir+lopinavir-ritonavir, favipiravir+placebo, lopinavir-ritonavir+placebo, and placebo-only arms, we recruited 61, 59, 60, and 60 participants and analysed 55, 56, 55, and 58 participants, respectively, who provided viral load measures at Day 1 and Day 5. In the primary analysis, the mean viral load in the favipiravir+placebo arm had changed by -0.57 log10 (95% CI -1.21 to 0.07, p = 0.08) and in the lopinavir-ritonavir+placebo arm by -0.18 log10 (95% CI -0.82 to 0.46, p = 0.58) compared to the placebo arm at Day 5. There was no significant interaction between favipiravir and lopinavir-ritonavir (interaction coefficient term: 0.59 log10, 95% CI -0.32 to 1.50, p = 0.20). More participants had undetectable virus at Day 5 in the favipiravir+placebo arm compared to placebo only (46.3% versus 26.9%, odds ratio (OR): 2.47, 95% CI 1.08 to 5.65; p = 0.03). Adverse events were observed more frequently with lopinavir-ritonavir, mainly gastrointestinal disturbance. Favipiravir drug levels were lower in the combination arm than the favipiravir monotherapy arm, possibly due to poor absorption. The major limitation was that the study population was relatively young and healthy compared to those most affected by the COVID-19 pandemic. CONCLUSIONS: At the current doses, no treatment significantly reduced viral load in the primary analysis. Favipiravir requires further evaluation with consideration of dose escalation. Lopinavir-ritonavir administration was associated with lower plasma favipiravir concentrations. TRIAL REGISTRATION: Clinicaltrials.gov NCT04499677 EudraCT: 2020-002106-68.

A large population sample of African HIV genomes from the 1980s reveals a reduction in subtype D over time associated with propensity for CXCR4 tropism.

We present 109 near full-length HIV genomes amplified from blood serum samples obtained during early 1986 from across Uganda, which to our knowledge is the earliest and largest population sample from the initial phase of the HIV epidemic in Africa. Consensus sequences were made from paired-end Illumina reads with a target-capture approach to amplify HIV material following poor success with standard approaches. In comparisons with a smaller 'intermediate' genome dataset from 1998 to 1999 and a 'modern' genome dataset from 2007 to 2016, the proportion of subtype D was significantly higher initially, dropping from 67% (73/109), to 57% (26/46) to 17% (82/465) respectively (p < 0.0001). Subtype D has previously been shown to have a faster rate of disease progression than other subtypes in East African population studies, and to have a higher propensity to use the CXCR4 co-receptor ("X4 tropism"); associated with a decrease in time to AIDS. Here we find significant differences in predicted tropism between A1 and D subtypes in all three sample periods considered, which is particularly striking the 1986 sample: 66% (53/80) of subtype D env sequences were predicted to be X4 tropic compared with none of the 24 subtype A1. We also analysed the frequency of subtype in the envelope region of inter-subtype recombinants, and found that subtype A1 is over-represented in env, suggesting recombination and selection have acted to remove subtype D env from circulation. The reduction of subtype D frequency over three decades therefore appears to be a result of selective pressure against X4 tropism and its higher virulence. Lastly, we find a subtype D specific codon deletion at position 24 of the V3 loop, which may explain the higher propensity for subtype D to utilise X4 tropism.

Applications of the hollow-fibre infection model (HFIM) in viral infection studies.

Conventional cell culture systems involve growing cells in stationary cultures in the presence of growth medium containing various types of supplements. At confluency, the cells are divided and further expanded in new culture dishes. This passage from confluent monolayer to sparse cultures does not reflect normal physiological conditions and represents quite a drastic physiological change that may affect the natural cell physiobiology. Hollow-fibre bioreactors were in part developed to overcome these limitations and since their inception, they have widely been used in production of monoclonal antibodies and recombinant proteins. These bioreactors are increasingly used to study antibacterial drug effects via simulation of in vivo pharmacokinetic profiles. The use of the hollow-fibre infection model (HFIM) in viral infection studies is less well developed and in this review we have analysed and summarized the current available literature on the use of these bioreactors, with an emphasis on viruses. Our work has demonstrated that this system can be applied for viral expansion, studies of drug resistance mechanisms, and studies of pharmacokinetic/pharmacodynamic (PK/PD) of antiviral compounds. These platforms could therefore have great applications in large-scale vaccine development, and in studies of mechanisms driving antiviral resistance, since the HFIM could recapitulate the same resistance mechanisms and mutations observed in vivo in clinic. Furthermore, some dosage and spacing regimens evaluated in the HFIM system, as allowing maximal viral suppression, are in line with clinical practice and highlight this 'in vivo-like' system as a powerful tool for experimental validation of in vitro-predicted antiviral activities.

Deep sequencing of HIV-1 reveals extensive subtype variation and drug resistance after failure of first-line antiretroviral regimens in Nigeria.

BACKGROUND: Deep sequencing could improve understanding of HIV treatment failure and viral population dynamics. However, this tool is often inaccessible in low- and middle-income countries. OBJECTIVES: To determine the genetic patterns of resistance emerging in West African HIV-1 subtypes during first-line virological failure, and the implications for future antiretroviral options. PATIENTS AND METHODS: Participants were selected from a Nigerian cohort of people living with HIV who had failed first-line ART and subsequently switched to second-line therapy. Whole HIV-1 genome sequences were generated from first-line virological failure samples with Illumina MiSeq. Mutations detected at ≥2% frequency were analysed and compared by subtype. RESULTS: HIV-1 sequences were obtained from 101 participants (65% female, median age 30 years, median 32.9 months of nevirapine- or efavirenz-based ART). Thymidine analogue mutations (TAMs) were detected in 61%, other core NRTI mutations in 92% and NNRTI mutations in 99%. Minority variants (<20% frequency) comprised 18% of all mutations. K65R was more prevalent in CRF02_AG than G subtypes (33% versus 7%; P = 0.002), and ≥3 TAMs were more common in G than CRF02_AG (52% versus 24%; P = 0.004). Subtype G viruses also contained more RT cleavage site mutations. Cross-resistance to at least one of the newer NNRTIs, doravirine, etravirine or rilpivirine, was predicted in 81% of participants. CONCLUSIONS: Extensive drug resistance had accumulated in people with West African HIV-1 subtypes, prior to second-line ART. Deep sequencing significantly increased the detection of resistance-associated mutations. Caution should be used if considering newer-generation NNRTI agents in this setting.