Breakthrough SARS-CoV-2 Infections in the PROVENT Prevention Trial Were Not Associated With AZD7442 (Tixagevimab/Cilgavimab) Resistant Variants.

BACKGROUND: We report spike protein-based lineage and AZD7442 (tixagevimab/cilgavimab) neutralizing activity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants identified from breakthrough infections in the PROVENT preexposure prophylaxis trial. METHODS: Variants identified from PROVENT participants with reverse-transcription polymerase chain reaction-positive symptomatic illness were phenotypically assessed to determine neutralization susceptibility of variant-specific pseudotyped virus-like particles. RESULTS: At completion of 6 months' follow-up, no AZD7442-resistant variants were observed in breakthrough coronavirus disease 2019 (COVID-19) cases. SARS-CoV-2 neutralizing antibody titers were similar in breakthrough and nonbreakthrough cases. CONCLUSIONS: Symptomatic COVID-19 breakthrough cases in PROVENT were not due to resistance-associated substitutions in AZD7442 binding sites or lack of AZD7442 exposure. CLINICAL TRIALS REGISTRATION: NCT04625725.

Induction of resistance to sugarcane mosaic virus by RNA interference targeting coat protein gene silencing in transgenic sugarcane.

Sugarcane mosaic virus (SCMV) is a serious disease of monocotyledonous plants, including sugarcane, causing deterioration in both growth and productivity. RNA interference (RNAi) inhibits gene expression through RNA-mediated sequence-specific interactions and is considered an effective approach to control viral infection in plants. In this study, the SCMVCp gene encoding the coat protein (CP) was inserted into the pGreen-0179 plasmid in both sense and antisense orientations. Cauliflower mosaic virus (CaMV) and Zea mays ubiquitin (Ubi) promoters were selected to drive the transcription of the intron-hairpin constructs, called HpSCMVCp-CaMV and HpSCMVCp-Ubi, respectively. Transgenic sugarcane expressing these constructs was generated through Agrobacterium-mediated transformation. This transformation method produced a high percentage of transgenic plants for both HpSCMVCp-CaMV and HpSCMVCp-Ubi, as confirmed by PCR analysis. Southern blotting revealed a single stable insertion of the DNA target in the genome of transgenic sugarcane lines. After artificial virus infection, lines that developed mosaic symptoms were classified as susceptible, whereas those that remained green without symptoms were classified as resistant at 42 days post-inoculation. Immunoblotting revealed CP expression at 37 kDa in susceptible and non-transgenic sugarcane, but not in resistant lines. RT-PCR analysis confirmed viral Cp and Nib gene expression in susceptible lines and their absence in resistant lines. Interestingly, upon comparison of effectivity, CaMV and Ubi promoter-driven gene expression resulted in 57.69% and 82.35% resistant sugarcane lines, respectively. Thus, we concluded that RNAi is effective for inducing resistance against SCMV and that the Ubi promoter is an effective promoter for producing transgenic sugarcane.

Functional Mapping of Regions Involved in the Negative Imprinting of Virion Particle Infectivity and in Target Cell Protection by Interferon-Induced Transmembrane Protein 3 against HIV-1.

The interferon-induced transmembrane proteins (IFITMs) are a family of highly related antiviral factors that affect numerous viruses at two steps: in target cells by sequestering incoming viruses in endosomes and in producing cells by leading to the production of virions that package IFITMs and exhibit decreased infectivity. While most studies have focused on the former, little is known about the regulation of the negative imprinting of virion particle infectivity by IFITMs and about its relationship with target cell protection. Using a panel of IFITM3 mutants against HIV-1, we have explored these issues as well as others related to the biology of IFITM3, in particular virion packaging, stability, the relation to CD63/multivesicular bodies (MVBs), the modulation of cholesterol levels, and the relationship between negative imprinting of virions and target cell protection. The results that we have obtained exclude a role for cholesterol and indicate that CD63 accumulation does not directly relate to an antiviral behavior. We have defined regions that modulate the two antiviral properties of IFITM3 as well as novel domains that modulate protein stability and that, in so doing, influence the extent of its packaging into virions. The results that we have obtained, however, indicate that, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for negative imprinting. Finally, while most mutations concomitantly affect target cell protection and negative imprinting, a region in the C-terminal domain (CTD) exhibits a differential behavior, potentially highlighting the regulatory role that this domain may play in the two antiviral activities of IFITM3.IMPORTANCE IFITM proteins have been associated with the sequestration of incoming virions in endosomes (target cell protection) and with the production of virion particles that incorporate IFITMs and exhibit decreased infectivity (negative imprinting of virion infectivity). How the latter is regulated and whether these two antiviral properties are related remain unknown. By examining the behavior of a large panel of IFITM3 mutants against HIV-1, we determined that IFITM3 mutants are essentially packaged into virions proportionally to their intracellular levels of expression. However, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for the antiviral effects. Most mutations were found to concomitantly affect both antiviral properties of IFITM3, but one CTD mutant exhibited a divergent behavior, possibly highlighting a novel regulatory role for this domain. These findings thus advance our comprehension of how this class of broad antiviral restriction factors acts.

Characterization of resistance to a potent D-peptide HIV entry inhibitor.

BACKGROUND: PIE12-trimer is a highly potent D-peptide HIV-1 entry inhibitor that broadly targets group M isolates. It specifically binds the three identical conserved hydrophobic pockets at the base of the gp41 N-trimer with sub-femtomolar affinity. This extremely high affinity for the transiently exposed gp41 trimer provides a reserve of binding energy (resistance capacitor) to prevent the viral resistance pathway of stepwise accumulation of modest affinity-disrupting mutations. Such modest mutations would not affect PIE12-trimer potency and therefore not confer a selective advantage. Viral passaging in the presence of escalating PIE12-trimer concentrations ultimately selected for PIE12-trimer resistant populations, but required an extremely extended timeframe (> 1 year) in comparison to other entry inhibitors. Eventually, HIV developed resistance to PIE12-trimer by mutating Q577 in the gp41 pocket. RESULTS: Using deep sequence analysis, we identified three mutations at Q577 (R, N and K) in our two PIE12-trimer resistant pools. Each point mutant is capable of conferring the majority of PIE12-trimer resistance seen in the polyclonal pools. Surface plasmon resonance studies demonstrated substantial affinity loss between PIE12-trimer and the Q577R-mutated gp41 pocket. A high-resolution X-ray crystal structure of PIE12 bound to the Q577R pocket revealed the loss of two hydrogen bonds, the repositioning of neighboring residues, and a small decrease in buried surface area. The Q577 mutations in an NL4-3 backbone decreased viral growth rates. Fitness was ultimately rescued in resistant viral pools by a suite of compensatory mutations in gp120 and gp41, of which we identified seven candidates from our sequencing data. CONCLUSIONS: These data show that PIE12-trimer exhibits a high barrier to resistance, as extended passaging was required to develop resistant virus with normal growth rates. The primary resistance mutation, Q577R/N/K, found in the conserved gp41 pocket, substantially decreases inhibitor affinity but also damages viral fitness, and candidate compensatory mutations in gp160 have been identified.

Is the conquest of Hepatitis C imminent?

Hepatitis C virus represents a global pathogen of human health significance. In the space of less than three decades, we have witnessed the discovery of the virus, a growing understanding of the structure and biology of the viral-encoded proteins and their interaction with the host cell and the sequencing of the viral genome. Most importantly, we have moved from early therapeutic strategies aimed at crude boosting of host anti-viral immunity, limited by side effects and with poor response rates, to therapies that directly exploit our understanding of viral biology. In this review, we discuss the significance of the virus, its' discovery and outline the advances in the molecular characterisation of the virus, before setting these within the context of contemporary and emerging therapeutic strategies as well as viral resistance mechanisms.

High level of treatment failure and drug resistance to first-line antiretroviral therapies among HIV-infected children receiving decentralized care in Senegal.

BACKGROUND: In Senegal in 2015, an estimated 4800 children were living with HIV, with 1200 receiving ARV treatment, of whom half had follow-up care in decentralized sites outside Dakar. However, until now no studies have determined the efficacy of pediatric treatment in decentralized settings, even though the emergence of viral resistance, particularly among children in Africa, is a well-known phenomenon. This study aimed to assess the virological status of HIV-infected children in all decentralized facilities to help improve access to quality care. METHODS: A cross-sectional epidemiological and virological study was conducted in all of Senegal's regions, except Dakar, between March and June 2015 and sought to include all HIV-infected children and adolescents (0-19 years), treated or not with ARVs. Socio-demographic and clinical data and a blood sample on blotting paper were collected for children from treatment sites. Samples were routed on public transportation, assisted by a network of community health workers. A viral load (VL) assay was performed for each child, followed by genotyping when it exceeded 1000 copies/mL (3 log10). RESULTS: Of the 851 identified children, 666 (78%) were enrolled in the study. Half of the children were girls, and the average age was 8 years (6 months-19 years). Most of the children (96.7%) were infected with HIV-1, and 90% were treated with ART, primarily with AZT + 3TC + NVP/EFV therapeutic regimen. The median duration of time on ART was 21 months (1-129). VL was measured for 2% of children before this study. Almost two-thirds (64%) of the children are experiencing virological failure. Among them, there was resistance to at least one drug for 86.5% of cases. Also, 25% children presented resistance to one drug and 40% to two out of three. For nearly one-third of the children presenting resistance, none of the three drugs of the treatment was active. Factors associated with virological failure were male sex, follow-up by a generalist rather than a specialist, and treatment interruptions. CONCLUSIONS: We observed a high level of virological failure and a high percentage of viral resistance among children receiving health care in decentralized facilities in Senegal.

Precision mapping of N- and O-glycoproteins in viral resistant and susceptible strains of Bombyx mori.

Protein glycosylation plays important roles in protein structure, function, and immune recognition, among many other activities. One of the major roles of glycans and glycoconjugates on the cell surface is acting as the receptor for outside pathogens such as viruses. During the initial stage of viral replication, viruses interact with cell membrane receptors, which are in many cases glycoproteins. Identifying such glycoproteins is essential to understanding the mechanisms of viral infection, as well as developing antiviral strategies. Silkworm is an important economic insect as well as a model organism for molecular biology, yet current knowledge on its glycoproteome is far from complete due to both analytical challenges and perceived lack of importance. In this study, we performed a large-scale glycoproteomic survey for two silkworm Bombyx mori strains 306 and NB, which are susceptible and resistant to the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), respectively. More than 400 silkworm N- and O- glycoproteins were identified with high confidence, demonstrating that this organism employs extensive glycosylation. We mapped some glycoproteins only to the BmNPV susceptible or resistant strain, underlining the potential relationship between glycosylation and viral susceptibility. We predicted O-glycoproteins and O-glycan compositions for the first time for this organism. The variations in glycan site occupancy, as well as glycan diversity between the two silkworm strains, provide an insight into role of glycosylation in viral recognition and infection processes.

Efficacy, Safety, and Predictors of Direct-acting antivirals in Hepatitis C Virus Patients with Heterogeneous Liver Diseases.

Safety, efficacy, and predictor factors of sustained-virological-response after 24 weeks of new direct-acting antivirals were evaluated in hepatitis C virus patients with different stages of hepatic disease. 260 patients, median age 60 years, of whom 48.1% cirrhotics, 17.7% liver transplant recipients, and 45.7% naïve were treated with Sofosbuvir+Ribavirine, Sofosbuvir+Simeprevir±Ribavirine, Sofosbuvir+Daclatasvir± Ribavirine, Sofosbuvir+Ledispavir±Ribavirine, Ombitasvir/Paritaprevir/Ritonavir+Ribavirine and Ombitasvir/Paritaprevir/Ritonavir+Dasabuvir±Ribavirine. Therapy outcomes, hematochemical parameters, viral replication, genotype, and resistance-associated-mutations were analyzed retrospectively. Sustained virological response was 90.4% in the whole population, 83.2% in cirrhotics, 85% in patients with previous virological failure, 93.6% in patients >60 years, and 95.6% in liver transplant recipients. SVR24 for each drug regimen was 75% Sofosbuvir+Ribavirine, 80.4% Sofosbuvir+Simeprevir±Ribavirine, 94.3% Sofosbuvir+Daclatasvir±Ribavirine, 98.7% Sofosbuvir+Ledispavir±Ribavirine, 100% Ombitasvir/ Paritaprevir/Ritonavir+Ribavirine and Ombitasvir/Paritaprevir/Ritonavir+Dasabuvir±Ribavirine. The highest sustained virological response rates were obtained with genotype-1b (95.9%). Twenty-five patients, mostly cirrhotics or suffering from severe liver complications, manifested relapse (84%), breakthrough (12%), or non-response (4%). Mild side effects were observed in 41.1% of patients. Model-for-End-Liver- Disease score <10 and alanine aminotransferase ≤20 U/L at week 8 of therapy proved positive predictors of sustained virological response. Direct-acting antiviral therapy is efficacious and safe even in patients with advanced liver disease and/ or previous virological failure; Model-for-End-Liver-Disease <10 and alanine aminotransferase reduction during therapy were found to be reliable predicting markers of sustained-virological-response.

Clinical Laboratory Testing in the Era of Directly Acting Antiviral Therapies for Hepatitis C.

Directly acting antiviral (DAA) combination therapies for chronic hepatitis C virus (HCV) infection are highly effective, but treatment decisions remain complex. Laboratory testing is important to evaluate a range of viral, host, and pharmacological factors when considering HCV treatment, and patients must be monitored during and after therapy for safety and to assess the viral response. In this review, we discuss the laboratory tests relevant for the treatment of HCV infection in the era of DAA therapy, grouped according to viral and host factors.

[Treatment of chronic viral hepatitis C with direct acting antiviral agent: review].

HCV infection treatment regimens are viewed from positions of HCV life cycle and replication, effects of NS3/4A protease inhibitors and NS5A/NS5B inhibitors on HCV strain replication. Evolution of HCV treatment regimens from its discovery to modern DAA agents had led to substantial progress although drug resistance poses a new issue to be addressed.

Robust RNAi-mediated resistance to infection of seven potyvirids in soybean expressing an intron hairpin NIb RNA.

Viral pathogens, such as soybean mosaic virus (SMV), are a major constraint in soybean production and often cause significant yield loss and quality deterioration. Engineering resistance by RNAi-mediated gene silencing is a powerful strategy for controlling viral diseases. In this study, a 248-bp inverted repeat of the replicase (nuclear inclusion b, NIb) gene was isolated from the SMV SC3 strain, driven by the leaf-specific rbcS2 promoter from Phaseolus vulgaris, and introduced into soybean. The transgenic lines had significantly lower average disease indices (ranging from 2.14 to 12.35) than did the non-transformed (NT) control plants in three consecutive generations, exhibiting a stable and significantly enhanced resistance to the SMV SC3 strain under field conditions. Furthermore, seed mottling did not occur in transgenic seeds, whereas the NT plants produced ~90% mottled seeds. Virus resistance spectrum screening showed that the greenhouse-grown transgenic lines exhibited robust resistance to five SMV strains (SC3, SC7, SC15, SC18, and a recombinant SMV), bean common mosaic virus, and watermelon mosaic virus. Nevertheless, no significantly enhanced resistance to bean pod mottle virus (BPMV, Comovirus) was observed in the transgenic lines relative to their NT counterparts. Consistent with the results of resistance evaluation, the accumulation of each potyvirid (but not of BPMV) was significantly inhibited in the transgenic plants relative to the NT controls as confirmed by quantitative real-time (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). These results demonstrate that robust RNAi-mediated resistance to multiple potyvirids in soybean was conferred by expressing an intron hairpin SMV NIb RNA.

Antiviral Resistance of Splenocytes in Aged Mice.

We compared the susceptibility to viral infection of splenocytes, isolated from young versus old CBA mice, and evaluated the antiviral actions of lactoferrin in splenocytes infected with Encephalomyocarditis virus (EMCV). Recombinant mouse lactoferrin (rmLF) and bovine lactoferrin (bLF) were used. There were no differences in the susceptibility to EMCV infection in the studied age categories. Both types of lactoferrins were protective in young and old mice. The study confirmed the undisturbed viral resistance in old mice and the protective actions of lactoferrin in viral infection. The antiviral action of the homologous mouse lactoferrin was demonstrated for the first time.

Resistance to hepatitis C virus. Implications and therapeutic options. / Resistencias al virus de la hepatitis C. Implicaciones y posibilidades terapéuticas.

We are currently living in an unprecedented era of hepatitis C treatment with the availability of highly effective drugs yielding minimal side effects. The problem we currently face is the retreatment of patients refractory to these drugs. Although several factors can influence treatment failure, this review focuses on antiviral resistance. Resistance-associated substitutions may be identified at baseline or be treatment-emergent. The latter seem to be more clinically relevant and must be studied in the event of treatment failure (no virological response). In this article, we present the latest data from clinical trials and studies in real-life clinical practice. Finally, based on this current evidence, we propose some recommendations for the management and retreatment of these patients.

Virus-induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two patterns of transgene body methylation.

We used bisulfite sequencing to study the methylation of a viral transgene whose expression was silenced upon plum pox virus infection of the transgenic plant and its subsequent recovery as a consequence of so-called virus-induced gene silencing (VIGS). VIGS was associated with a general increase in the accumulation of small RNAs corresponding to the coding region of the viral transgene. After VIGS, the transgene promoter was not methylated and the coding region showed uneven methylation, with the 5' end being mostly unmethylated in the recovered tissue or mainly methylated at CG sites in regenerated silenced plants. The methylation increased towards the 3' end, which showed dense methylation in all three contexts (CG, CHG and CHH). This methylation pattern and the corresponding silenced status were maintained after plant regeneration from recovered silenced tissue and did not spread into the promoter region, but were not inherited in the sexual offspring. Instead, a new pattern of methylation was observed in the progeny plants consisting of disappearance of the CHH methylation, similar CHG methylation at the 3' end, and an overall increase in CG methylation in the 5' end. The latter epigenetic state was inherited over several generations and did not correlate with transgene silencing and hence virus resistance. These results suggest that the widespread CG methylation pattern found in body gene bodies located in euchromatic regions of plant genomes may reflect an older silencing event, and most likely these genes are no longer silenced.

Feasibility, safety, efficacy and potential scaling-up of sofosbuvir-based HCV treatment in Central and West Africa: (TAC ANRS 12311 trial).

Access to Hepatis C treatment in Sub-Saharan Africa is a clinical, public health and ethical concern. The multi-country open-label trial TAC ANRS 12311 allowed assessing the feasibility, safety, efficacy of a specific care model of HCV treatment and retreatment in patients with hepatitis C in Sub Saharan Africa. Between November 2015 and March 2017, with follow-up until mid 2019, treatment-naïve patients with HCV without decompensated cirrhosis or liver cancer were recruited to receive 12 week-treatment with either sofosbuvir + ribavirin (HCV genotype 2) or sofosbuvir + ledipasvir (genotype 1 or 4) and retreatment with sofosbuvir + velpatasvir + voxilaprevir in case of virological failure. The primary outcome was sustained virological response at 12 weeks after end of treatment (SVR12). Secondary outcomes included treatment adherence, safety and SVR12 in patients who were retreated due to non-response to first-line treatment. The model of care relied on both viral load assessment and educational sessions to increase patient awareness, adherence and health literacy. The study recruited 120 participants, 36 HIV-co-infected, and 14 cirrhotic. Only one patient discontinued treatment because of return to home country. Neither death nor severe adverse event occurred. SVR12 was reached in 107 patients (89%): (90%) in genotype 1 or 2, and 88% in GT-4. All retreated patients (n = 13) reached SVR12. HCV treatment is highly acceptable, safe and effective under this model of care. Implementation research is now needed to scale up point-of-care HCV testing and SVR assessment, along with community involvement in patient education, to achieve HCV elimination in Sub-Saharan Africa.

An in vitro experimental pipeline to characterize the epitope of a SARS-CoV-2 neutralizing antibody.

IMPORTANCE: The COVID-19 pandemic remains a significant public health concern for the global population; the development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread so long as they recognize and interact with circulating variants. The epitope and binding specificity of a neutralizing anti-SARS-CoV-2 Spike receptor-binding domain antibody clone against many SARS-CoV-2 variants of concern were characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis and VSV-spike neutralization studies. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

Strategies for Engineering Virus Resistance in Potato.

Potato (Solanum tuberosum L.) is an important vegetable crop that plays a pivotal role in the world, especially given its potential to feed the world population and to act as the major staple food in many developing countries. Every year, significant crop loss is caused by viral diseases due to a lack of effective agrochemical treatments, since only transmission by insect vectors can be combated with the use of insecticides, and this has been an important factor hindering potato production. With the rapid development of molecular biology and plant genetic engineering technology, transgenic approaches and non-transgenic techniques (RNA interference and CRISPR-cas9) have been effectively employed to improve potato protection against devastating viruses. Moreover, the availability of viral sequences, potato genome sequences, and host immune mechanisms has remarkably facilitated potato genetic engineering. In this study, we summarize the progress of antiviral strategies applied in potato through engineering either virus-derived or plant-derived genes. These recent molecular insights into engineering approaches provide the necessary framework to develop viral resistance in potato in order to provide durable and broad-spectrum protection against important viral diseases of solanaceous crops.

Synergistic inhibition of hepatitis C virus infection by a novel microtubule inhibitor in combination with daclatasvir.

Even though substantial progress has been made in the treatment of hepatitis C virus (HCV) infection, viral resistance and relapse still occur in some patients and additional therapeutic approaches may ultimately be needed should viral resistance become more prevalent. Microtubules play important roles in several HCV life cycle events, including cell attachment, entry, cellular transportation, morphogenesis and progeny secretion steps. Therefore, it was hypothesized that microtubular inhibition might be a novel approach for the treatment of HCV infection. Here, the inhibitory effects of our recently developed microtubule inhibitors were studied in the HCV replicon luciferase reporter system and the infectious system. In addition, the combination responses of microtubule inhibitors with daclatasvir, which is a clinically used HCV NS5A inhibitor, were also evaluated. Our results indicated that microtubule targeting had activity against HCV replication and showed synergistic effect with a current clinical drug.