Replicative Capacity Research Articles

Earlier In Vitro Viral Production With SARS-CoV-2 Alpha Than With Beta, Gamma, B, or A.27 Variants.

Since its emergence in China at the end of 2019, SARS-CoV-2 has rapidly spread across the world to become a global public health emergency. Since then, the pandemic has evolved with the large worldwide emergence of new variants, such as the Alpha (B.1.1.7 variant), Beta (B.1.351 variant), and Gamma (P.1 variant), and some other under investigation such as the A.27 in France. Many studies are focusing on antibody neutralisation changes according to the spike mutations, but to date, little is known regarding their respective replication capacities. In this work, we demonstrate that the Alpha variant provides an earlier replication in vitro, on Vero E6 and A549 cells, than Beta, Gamma, A.27, and historical lineages. This earlier replication was associated with higher infectious titres in cell-culture supernatants, in line with the higher viral loads observed among Alpha-infected patients. Interestingly, Beta and Gamma variants presented similar kinetic and viral load than the other non-Alpha-tested variants.

Molecular Evolution of Infectious Bronchitis Virus and the Emergence of Variant Viruses Circulating in the United States.

Infectious bronchitis virus (IBV) is a highly infectious and transmissible gammacoronavirus that is nearly impossible to control through biosecurity. Coronaviruses are RNA viruses with an enormous capacity for rapid replication and high rates of mutation, leading to a tremendous amount of genetic diversity. Viral evolution occurs when selection working on genetic diversity leads to new mutations being fixed in the population over time. For IBV, the emergence of variant viruses is likely due to a combination of selection acting on existing genetic diversity, as well as on newly created mutations as the virus replicates, or genetic drift. Immunity against IBV creates a strong selection pressure; however, immunity can also reduce the viral load, decreasing replication and the development of new mutations. Examining the balance between immunity reducing infection, replication, and genetic diversity, and immune pressure selecting for new variants, is extremely difficult at best. Nonetheless, vaccination and immunity do play a role in the emergence of new antigenic variants of IBV. To complicate the situation even more, coronaviruses can undergo recombination, and several studies in the literature report recombination between IBV vaccines and field viruses. However, to our knowledge, unlike genetic drift, recombination alone has not been shown to result in a new antigenic and pathogenic IBV type emerging to cause widespread disease in poultry. Vaccines against IBV that result in an immune population can reduce transmission (basic reproductive number R0 less than 1), making vaccines for IBV the best control strategy available. However, IBV control remains extremely challenging because of the high number of antigenic variants causing disease in poultry and a limited number of vaccines that mostly provide only partial protection against infection and replication of those variants. Currently, there is one major variant IBV circulating in all sectors of US commercial poultry production: DMV/1639/11. This virus was initially detected in 2011, but only began causing significant disease in 2014/2015. Since then, it has affected all three sectors of poultry production (layers, breeders, broilers) and continues to predominate in certain regions of the United States. Additionally, a previously classified variant IBV, which is no longer considered a variant virus, GA08, is highly prevalent. This is attributed to heavy GA08-type IBV vaccine usage because disease caused by the GA08-type virus is rare. Interestingly, the major IBV detected in poultry for several decades, ArkDPI, is no longer among the most detected viruses in the United States. This change corresponds to the shift away from ArkDPI vaccine usage in the broiler sector as GA08 vaccine usage has increased and highlights the role IBV vaccines play in influencing viral populations in commercial chickens.

H9N2 virus-derived M1 protein promotes H5N6 virus release in mammalian cells: Mechanism of avian influenza virus inter-species infection in humans.

H5N6 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4 not only exhibits unprecedented intercontinental spread in poultry, but can also cause serious infection in humans, posing a public health threat. Phylogenetic analyses show that 40% (8/20) of H5N6 viruses that infected humans carried H9N2 virus-derived internal genes. However, the precise contribution of H9N2 virus-derived internal genes to H5N6 virus infection in humans is unclear. Here, we report on the functional contribution of the H9N2 virus-derived matrix protein 1 (M1) to enhanced H5N6 virus replication capacity in mammalian cells. Unlike H5N1 virus-derived M1 protein, H9N2 virus-derived M1 protein showed high binding affinity for H5N6 hemagglutinin (HA) protein and increased viral progeny particle release in different mammalian cell lines. Human host factor, G protein subunit beta 1 (GNB1), exhibited strong binding to H9N2 virus-derived M1 protein to facilitate M1 transport to budding sites at the cell membrane. GNB1 knockdown inhibited the interaction between H9N2 virus-derived M1 and HA protein, and reduced influenza virus-like particles (VLPs) release. Our findings indicate that H9N2 virus-derived M1 protein promotes avian H5N6 influenza virus release from mammalian, in particular human cells, which could be a major viral factor for H5N6 virus cross-species infection.

Identification of a novel replication-competent hepatitis C virus variant that confers the sofosbuvir resistance

Despite the excellent antiviral potency of direct-acting antivirals (DAAs) against hepatitis C virus (HCV), emergence of drug-resistant viral mutations remains a potential challenge. Sofobuvir (SOF), a nucleotide analog targeting HCV NS5B - RNA-dependent RNA polymerase (RdRp), constitutes a key component of many anti-HCV cocktail regimens and confers a high barrier for developing drug resistance. The serine to threonine mutation at the amino acid position 282 of NS5B (S282T) is the mostly documented SOF resistance-associated substitution (RAS), but severely hampers the virus fitness. In this study, we first developed new genotype 1b (GT1b) subgenomic replicon cells, denoted PR52D4 and PR52D9, directly from a GT1b clinical isolate. Next, we obtained SOF-resistant and replication-competent PR52D4 replicon by culturing the replicon cells in the presence of SOF. Sequencing analysis showed that the selected replicon harbored two mutations K74R and S282T in NS5B. Reverse genetics analysis showed that while PR52D4 consisting of either single mutation K74R or S282T could not replicate efficiently, the engineering of the both mutations led to a replication-competent and SOF-resistant PR52D4 replicon. Furthermore, we showed that the K74R mutation could also rescue the replication deficiency of the S282T mutation in Con1, another GT1b replicon as well as in JFH1, a GT2a replicon. Structural modeling analysis suggested that K74R might help maintain an active catalytic conformation of S282T by engaging with Y296. In conclusion, we identified the combination of two NS5B mutations S282T and K74R as a novel RAS that confers a substantial resistance to SOF while retains the HCV replication capacity.

Real-world efficacy and safety of direct-acting antiviral drugs in patients with chronic hepatitis C and inherited blood disorders.

BackgroundPatients with inherited blood disorders (IBLD) have a high risk of hepatitis C virus (HCV) infection. The aim of this work was to assess the efficacy and safety of HCV direct-acting antiviral (DAA)-based treatment in patients with IBLD and chronic HCV infection.MethodsTwenty-seven patients (25 with sickle cell disease, 1 with β-thalassemia and 1 with hemoglobin D-Punjab), including 3 with compensated cirrhosis, were included. They were treated with sofosbuvir in combination with ribavirin, daclatasvir, ledipasvir, or velpatasvir or with grazoprevir/elbasvir for 8 or 12 weeks. In the case of treatment failure, in-vitro assessment of resistance-associated substitutions (RASs) and full-length genome sequence analysis by means of deep sequencing were performed.Results:Treatment was safe and well-tolerated and there were no drug discontinuations due to DAA-related adverse events. Twenty-five out of the 27 patients (93%) achieved sustained virological response 12 weeks post-treatment. One patient discontinued after 18 days due to adverse events unrelated to the antiviral treatment. One patient infected with ‘unusual’ genotype 2 subtype 2m relapsed. Subtype 2m naturally carries the NS5A L31M RAS. In a genotype 2a subgenomic replicon model, L31M increased daclatasvir effective concentration 50% (EC50) by 97-fold, but velpatasvir EC50 by only 3-fold, without altering the replication capacity. This patient was successfully retreated with sofosbuvir/velpatasvir for 12 weeks.ConclusionDAA-based regimens are well tolerated and highly efficacious in patients with chronic hepatitis C and IBLD in the real-world setting. Thus, DAA-based antiviral treatment should be prioritized in this thus far neglected population of HCV-infected patients.

In vivo Serial Passaging of Human-Simian Immunodeficiency Virus Clones Identifies Characteristics for Persistent Viral Replication.

We previously reported that a human immunodeficiency virus type 1 with a simian immunodeficiency virus vif substitution (HSIV-vifNL4-3) could replicate in pigtailed macaques (PTMs), demonstrating that Vif is a species-specific tropism factor of primate lentiviruses. However, infections did not result in high-peak viremia or setpoint plasma viral loads, as observed during simian immunodeficiency virus (SIV) infection of PTMs. Here, we characterized variants isolated from one of the original infected animals with CD4 depletion after nearly 4years of infection to identify determinants of increased replication fitness. In our studies, we found that the HSIV-vif clones did not express the HIV-1 Vpr protein due to interference from the vpx open reading frame (ORF) in singly spliced vpr mRNA. To examine whether these viral genes contribute to persistent viral replication, we generated infectious HSIV-vif clones expressing either the HIV-1 Vpr or SIV Vpx protein. And then to determine viral fitness determinants of HSIV-vif, we conducted three rounds of serial in vivo passaging in PTMs, starting with an initial inoculum containing a mixture of CXCR4-tropic [Vpr-HSIV-vifNL4-3 isolated at 196 (C/196) and 200 (C/200) weeks post-infection from a PTM with depressed CD4 counts] and CCR5-tropic HSIV (Vpr+ HSIV-vif derivatives based NL-AD8 and Bru-Yu2 and a Vpx expressing HSIV-vifYu2). Interestingly, all infected PTMs showed peak plasma viremia close to or above 105 copies/ml and persistent viral replication for more than 20weeks. Infectious molecular clones (IMCs) recovered from the passage 3 PTM (HSIV-P3 IMCs) included mutations required for HIV-1 Vpr expression and those mutations encoded by the CXCR4-tropic HSIV-vifNL4-3 isolate C/196. The data indicate that the viruses selected during long-term infection acquired HIV-1 Vpr expression, suggesting the importance of Vpr for in vivo pathogenesis. Further passaging of HSIV-P3 IMCs in vivo may generate pathogenic variants with higher replication capacity, which will be a valuable resource as challenge virus in vaccine and cure studies.

Abstract 11757: Telomerase Treatment Restores Transcriptional Profile and Epigenetic Landscape in Progeria Vascular Cells

Hutchinson-Gilford progeria syndrome (HGPS) is a model of accelerated vascular aging caused by a de novo mutation of a single nucleotide in the Lamin A gene. The accumulation of abnormal lamin A (progerin) in the nuclear envelope induces a nuclear dysmorphology associated with global gene dysregulation, increased expression of inflammatory cytokines, impaired vascular function, and accelerated telomere erosion. These individuals succumb to myocardial infarction or stroke in their mid-teens. We hypothesized that restoration of telomere length might reverse some of the observed derangements in cell function, transcriptional profile and epigenetic alterations. Accordingly, we generated iPSC-derived endothelial cells (ECs) from HGPS children and their unaffected parents. By comparison to the parental controls, HGPS ECs have severely impaired replicative capacity, cellular functions (e.g. NO synthesis, acLDL uptake, network formation, cytokine secretion) and severe telomere shortening. We observed that transient transfection with mRNA encoding human telomerase (hTERT) increases telomere length, restores cellular replicative capacity and improves HGPS-EC functions. Notably, RNAseq studies revealed that that approximately 1200 genes were dysregulated in Progeria, most of which were improved by hTERT treatment, among which approximately 250+ genes were fully normalized. Intriguingly, hTERT treatment reduces DNA damage signals (γH2Ax) and reverses the aberrant chromatin accessibility observed in HGPS-ECs, as assessed by a reduction in the activating histone mark H3K4me3; an increase in the suppressive mark H3K4me3; and normalizes the level of heterochromatin markers HP1, Histone H1 and H3. Preliminary results from ChIPseq studies targeting histone marks characteristic of activators, repressors and enhancers as well as chromatin remodeling proteins; together with Hi-C analyses indicate that there are global changes in topologically associated domains (TADs) and other features of the chromatin landscape in HGPS ECs, which are partially restored by hTERT treatment. We conclude that hTERT treatment has surprisingly global effects on transcriptional profile and epigenetic landscape in addition to telomere extension.

Abstract 11821: Telomerase Therapy Reverses Vascular Senescence and Extends Lifespan in Progeria Mice

Background: Hutchinson-Gilford Progeria Syndrome (HGPS) is an accelerated aging syndrome associated with premature vascular disease and death due to heart attack and stroke. In HGPS a mutation in lamin A (progerin), alters nuclear morphology and gene expression. Current therapy increases the lifespan of these children only modestly. Thus, greater understanding of the underlying mechanisms of HGPS is required to improve therapy. Endothelial cells (ECs) differentiated from induced pluripotent stem cells (iPSCs) derived from these patients exhibit hallmarks of senescence including replication arrest, DNA damage, increased expression of inflammatory markers and telomere erosion. We hypothesized that correction of shortened telomeres may reverse these measures of vascular aging. Methods: We generated ECs from iPSCs belong to children with HGPS and their unaffected parents. Telomerase mRNA (hTERT) was used to treat HGPS ECs. Endothelial morphology and functions were assessed, as well as proteomic and transcriptional profiles with attention to inflammatory markers, DNA damage and EC identity genes. In a mouse model of HGPS, we assessed the effects of lentiviral transfection of mTERT on measures of senescence, focusing on the EC phenotype in various organs. Results: hTERT treatment of human HGPS ECs improved replicative capacity; restored endothelial functions such as nitric oxide generation, acLDL uptake and angiogenesis; and reduced the elaboration of inflammatory cytokines. In addition, hTERT treatment improved cellular and nuclear morphology, in association with a normalization of the transcriptional profile, effects which may be mediated in part by a reduction in progerin expression and an increase in SIRT1. Progeria mice treated with mTERT Lentivirus manifested similar improvements, with a reduction in inflammatory and DNA damage markers and increased SIRT1 in their vasculature and other organs. Furthermore, mTERT therapy increased the lifespan of HGPS mice. Conclusion: Vascular rejuvenation using telomerase mRNA is a promising approach for progeria and other age-related diseases.

In vitro analysis of the replicative capacity and phenotypic susceptibility to integrase inhibitors of HIV-2 mutants with integrase insertions.

HIV-2 resistance to integrase strand-transfer inhibitors (INSTIs) is characterized by two main pathways: (i) mutations at codons 143, 148 and155; and (ii) amino acid insertion after integrase codon 231 (231ins). To complete INSTI resistance data on HIV-2 by determining the viral replicative capacity and INSTI phenotypic susceptibility of integrase mutants obtained through site-directed mutagenesis. Site-directed mutants (SDMs) were constructed and viral stocks produced. Viral replicative capacity was assessed by measuring HIV-2 viral load at days 3, 7 and 14. In vitro phenotypic susceptibility was measured using the ANRS PBMC assay. Viruses bearing 231ins did not present impaired replicative capacity, except the 231ins GIRGK mutant. A 231ins GK SDM was resistant to raltegravir and cabotegravir, but remained susceptible to dolutegravir and bictegravir. SDMs harbouring a 5 amino acid insertion (GYKGK or SREGK) were both resistant to all INSTIs. The SDM with T97A+N155H, with or without E92Q, was resistant to all INSTIs, except bictegravir. These first data on the newly described resistance pathway 231ins, using site-directed mutagenesis, showed no measurable impact on viral fitness and confirmed the decreased susceptibility to a first-generation INSTI (raltegravir) and cabotegravir. Resistance to second-generation INSTIs (dolutegravir and bictegravir) occurred for mutants with a 5 amino acid 231ins.

Development of Multi-Engineered iPSC-Derived CAR-NK Cells for the Treatment of B-Cell Malignancies

Development of Multi-Engineered iPSC-Derived CAR-NK Cells for the Treatment of B-Cell Malignancies

Prognostic Value of Dynamic Monoclonal Protein Pattern on Probability of Myeloma Progression from the Precursor State

Multiple Myeloma (MM) is a cancer of terminally differentiated plasma cell resides in bone marrow, which is always preceded by clinically asymptomatic precursor states. The process of malignant transformation however is not fully understood. Analyses of cells from precursor state have provided evidence that it is a genetically advanced lesion, wherein tumor cells carry many of the genetic changes found in MM cells. Furthermore, mice xenografts from patient (pt) with precursor disease showed progressive growth on its own suggesting progression potential is counteracted by active extrinsic restraints that prevent its progression to MM (Das R et al. Nature Medicine, 2016). Hence, precursor progression to MM can be viewed as a dynamic process in which the clonal mass is the net result of pro-growth, pro-survival replicative capacity of the myeloma clones vs. anti-tumor immunity.The change in the net clonal mass sometimes is large enough to be detected by measurable fluctuations in serum monoclonal protein spike (M-spike) levels and reflects a highly dynamic battle between plasma cell clone and cell-mediated anti-myeloma immunity among those pts. The prognostic value of this highly dynamic state on risk of progression to MM is largely unknown. In this study we sought to assess the association between the dynamic M-spike pattern and the risk of progression from a precursor clone to MM.Methods: All pts with positive serum electrophoresis (SPEP) values measured from January 2011 to January 2021 for our institution were included for this study. All pts that had <3 M-spike readings before requiring anti-myeloma regimen, received active treatment for MM within 90 days of the first positive SPEP, had M-spike values >3g/l or had clear evidence of progression by serial increasing M-spike values were excluded.. We assigned the pattern of M-spike values over time into two groups according to stability. To eliminate the test-to-test variability, we defined stable disease as lack of more than 20% difference from lowest M-spike, and fluctuating disease if the difference exceeds 20%. The time-to-treatment (TTT) was then measured from the first positive SPEP to the time of treatment and was censored at the date of last follow-up for pts who did not have anti-myeloma treatment with death as competing risk. The effect of M-spike pattern on TTT, treating as binary outcome, was also evaluated using logistic regression analysis. Univariate analysis of TTT was performed using Gray's method. All tests were two-sided and p-value ≤ 0.05 were considered statistically significant.Results: A total of 565 pts were studied, 193 with a fluctuating pattern and 372 with a stable pattern. Median age at first positive SPEP was 76 years old (range: 38-101), 271 (48%) of them were male; 427 (75%) were Caucasian and 123 (22%) African American. Baseline creatinine was 1.17 mg/dl (range 0.5-11.2 SD 0.94). Baseline albumin was 3.97 g/dl (range 1.6-5.2 SD 0.47), 336 pts (59%) were IgG, 75 (13%) IgA, and 142 (25%) had IgM M-spike; 326 (58%) pts had Kappa light chain and 239 (42%) had Lambda. Autoimmune diseases were more common in the stable disease pattern (17% vs. 10% p=0.028), the most frequent one was Rheumatoid arthritis in 33 pts (6%). Furthermore, baseline albumin was statistically higher in pts in the stable cohort (4.04 vs. 3.85g/dl, p=<0.0001). Median follow up was 46.9 months (range: 3.2-120.9). Logistic regression: the odds ratio (OR) of having progression to treatment for fluctuation group was 4.5 (95% CI: 1.37-14.18, p= 0.013) in comparison to stable group. Time-to-event analysis: pts with fluctuating M-spike had higher chance of needing therapy (Hazard Ratio: 3.73, 95% CI: 1.19-11.66, p=0.024) than stable group. Cumulative incidence of needing treatment is shown in Figure-1.Conclusion: The dynamic pattern of M-spike seen here with fluctuating values over time (over time is two words) was significantly predictive of progression to MM in pts with a precursor disease state. These findings may reflect a different nature of tumor vs. immune system balance and possible impaired immunity in these pts which may have implications for future clinical trials that evaluate the efficacy of anti-myeloma immunotherapeutics. Furthermore, our finding suggest that fluctuations in M-spike levels may be factored into risk models that predict the progression to MM among pts with precursor clones. Larger studies are warranted to further study this concept. [Display omitted] DisclosuresMalek: Medpacto Inc.: Research Funding; Janssen: Other: Advisory board ; Takeda: Honoraria; Cumberland Inc.: Research Funding; Amgen: Honoraria; BMS: Honoraria, Research Funding; Bluespark Inc.: Research Funding; Sanofi: Other: Advisory Board.

Biological Characterization of Hepatitis B virus Genotypes: Their Role in Viral Replication and Antigen Expression.

Hepatitis B virus (HBV) inter-host evolution has resulted in genomic diversification reflected in the existence of nine genotypes (A-I) and numerous subgenotypes. There is growing evidence that genotypes influence HBV natural history, clinical outcomes, and treatment response. However, the biological characteristics underlying these differences have not yet been established. By transfecting HuH-7 cells with unit-length constructs of genotypes A2, B2, C1, D1, and F1b, we identified major differences in HBV replicative capacity and antigen expression across genotypes. Genotypes B2 and F1b showed a 2-fold increase in cccDNA levels compared to the other genotypes (p<0.005). Genotype A2 expressed the lowest pgRNA levels, with a 70-fold decrease in relation to the other genotypes (p<0.0001), while genotype B2 showed the lowest Precore RNA levels, with a 100-fold reduction compared to genotype A2 (p<0.0001). The highest intracellular HBV DNA levels were observed for genotype B2 and the lowest for genotypes A2 and C1 (p<0.0001). Regarding antigen expression, genotype F1b secreted the highest HBsAg levels and genotype D1 the lowest (p<0.0001), while genotypes A2 and B2 showed the highest intracellular HBsAg levels (p<0.0001). Interestingly, genotype C1 secreted the highest HBeAg levels, while genotype A2 showed the highest intracellular levels (p<0.0001). Finally, the analysis of the intra/extracellular antigen ratios revealed that most genotypes retained intracellularly 5–20% of the antigens, except the genotype A2 that retained 50% of the total expressed antigens. In conclusion, this study provides new insights into the biological characteristics of HBV genotypes, being the first study to comparatively analyze European (A and D) and Asian (B and C) genotypes with the Latin American (F) genotype. The differences in HBV replication and antigen expression might contribute to understand the differential role of genotypes in pathogenesis.

High Mutation Rate Leads to Fitness Loss for Coronavirus Quasispecies

Background : RNA viruses evolve very fast, with a mutation rate of 103 to 105 base substitution per nucleotides per copy. The mutation is a survival strategy for the viruses, which leads them to survive in the new host. Fitness is defined as the replication capacity of the virus in an experimental setup. Generally, the large population passage of the virus leads to fitness gain, but the world data of the coronavirus infection and death shows the flattened curve with time. It is contradictory to the principle of fitness gain due to large population passage. The coronavirus is losing its potency but remains infectious as it is passaging into millions that leads to a decline in the death of COVID patients and high recovery rates. Fitness loss of coronaviruses attributed to a high level of mutation in the RNA genome as well as host immune response. The current outbreak of SARS CoV-2 is surfaced in December 2019 in Hubei province of China and considered as bats/pangolin origin, spreading 235 countries of the world, infecting nearly 31,664,104 people, and claimed nearly 972,221 lives as of September 24, 2020 (Death rate approximately 3%). This coronavirus has passaged into 31,664,104 people from the beginning of this pandemic until September 24, 2020. Now the virus is losing potency rather than being monotonous and continuous in producing virusrelated complications. The population is still getting infected at the same rate, but the severity of the disease is reduced due to the potency of the virus diminished due to the passage effect as well as fitness loss of the virus due to high mutation rates. The death rate is reduced to 3% as compared to 6% in June 2020, when this paper was first submitted. Objective: The purpose of the study is to prove the fact that the coronavirus loses its potency with time but, they remain infective. It becomes more infectious due to mutation of the gene but loses the capacity to kill the host. Methods: Since the WHO announces the COVID-19 outbreak is an emergency of international concern, every country in the world is taking many measures to mitigate the viral load to their population. Simultaneously, the WHO, CDC USA, CDC Europe, and much other organization is updating the COVID cases and death online daily as reported by the respective country. With the help of the COVID-19 outbreak data published by the European CDC and ourworldindata.org, we correlate the total cases of coronavirus and total death in the top ten affected countries in the world. We also link the trends of total cases vs. total death and total new cases vs. total new death related to COVID-19 in Germany, Spain, the United Kingdom, Italy, and New Zealand from January 30, 2020, until September 24, 2020. The reason to select these countries for the study is that these countries updating the COVID cases and deaths regularly and said to achieve the peak of COVID related infections and recovering from the pandemic. Results: We have tried to correlate the high mutation rate of the virus that leads to losing its potency to severe infection and death in the human. Viral extinction through high mutation could be considered as the new anti-viral strategies. Conclusion: Coronavirus is losing its potency to causing death to the human. The new infection is still being reported from every corner of the world, but the death rate is significantly decreasing.

Residues T48 and A49 in HIV-1 NL4-3 Nef are responsible for the counteraction of autophagy initiation, which prevents the ubiquitin-dependent degradation of Gag through autophagosomes

BackgroundAutophagy plays an important role as a cellular defense mechanism against intracellular pathogens, like viruses. Specifically, autophagy orchestrates the recruitment of specialized cargo, including viral components needed for replication, for lysosomal degradation. In addition to this primary role, the cleavage of viral structures facilitates their association with pattern recognition receptors and MHC-I/II complexes, which assists in the modulation of innate and adaptive immune responses against these pathogens. Importantly, whereas autophagy restricts the replicative capacity of human immunodeficiency virus type 1 (HIV-1), this virus has evolved the gene nef to circumvent this process through the inhibition of early and late stages of the autophagy cascade. Despite recent advances, many details of the mutual antagonism between HIV-1 and autophagy still remain unknown. Here, we uncover the genetic determinants that drive the autophagy-mediated restriction of HIV-1 as well as the counteraction imposed by Nef. Additionally, we also examine the implications of autophagy antagonism in HIV-1 infectivity.ResultsWe found that sustained activation of autophagy potently inhibits HIV-1 replication through the degradation of HIV-1 Gag, and that this effect is more prominent for nef-deficient viruses. Gag re-localizes to autophagosomes where it interacts with the autophagosome markers LC3 and SQSTM1. Importantly, autophagy-mediated recognition and recruitment of Gag requires the myristoylation and ubiquitination of this virus protein, two post-translational modifications that are essential for Gag’s central role in virion assembly and budding. We also identified residues T48 and A49 in HIV-1 NL4-3 Nef as responsible for impairing the early stages of autophagy. Finally, a survey of pandemic HIV-1 transmitted/founder viruses revealed that these isolates are highly resistant to autophagy restriction.ConclusionsThis study provides evidence that autophagy antagonism is important for virus replication and suggests that the ability of Nef to counteract autophagy may have played an important role in mucosal transmission. Hence, disabling Nef in combination with the pharmacological manipulation of autophagy represents a promising strategy to prevent HIV spread.

Viral and Cellular Factors Leading to the Loss of CD4 Homeostasis in HIV-1 Viremic Nonprogressors.

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) viremic nonprogressors (VNPs) represent a very rare HIV-1 extreme phenotype. VNPs are characterized by persistent high plasma viremia and maintenance of CD4+ T-cell counts in the absence of treatment. However, the causes of nonpathogenic HIV-1 infection in VNPs remain elusive. Here, we identified for the first time two VNPs who experienced the loss of CD4+ homeostasis (LoH) after more than 13 years. We characterized in deep detail viral and host factors associated with the LoH and compared with standard VNPs and healthy controls. The viral factors determined included HIV-1 coreceptor usage and replicative capacity. Changes in CD4+ and CD8+ T-cell activation, maturational phenotype, and expression of CCR5 and CXCR6 in CD4+ T-cells were also evaluated as host-related factors. Consistently, we determined a switch in HIV-1 coreceptor use to CXCR4 concomitant with an increase in replicative capacity at the LoH for the two VNPs. Moreover, we delineated an increase in the frequency of HLA-DR+CD38+ CD4+ and CD8+ T cells and traced the augment of naive T-cells upon polyclonal activation with LoH. Remarkably, very low and stable levels of CCR5 and CXCR6 expression in CD4+ T-cells were measured over time. Overall, our results demonstrated HIV-1 evolution toward highly pathogenic CXCR4 strains in the context of very limited and stable expression of CCR5 and CXCR6 in CD4+ T cells as potential drivers of LoH in VNPs. These data bring novel insights into the correlates of nonpathogenic HIV-1 infection.IMPORTANCE The mechanism behind nonpathogenic human immunodeficiency virus type 1 (HIV-1) infection remains poorly understood, mainly because of the very low frequency of viremic nonprogressors (VNPs). Here, we report two cases of VNPs who experienced the loss of CD4+ T-cell homeostasis (LoH) after more than 13 years of HIV-1 infection. The deep characterization of viral and host factors supports the contribution of viral and host factors to the LoH in VNPs. Thus, HIV-1 evolution toward highly replicative CXCR4 strains together with changes in T-cell activation and maturational phenotypes were found. Moreover, we measured very low and stable levels of CCR5 and CXCR6 in CD4+ T-cells over time. These findings support viral evolution toward X4 strains limited by coreceptor expression to control HIV-1 pathogenesis and demonstrate the potential of host-dependent factors, yet to be fully elucidated in VNPs, to control HIV-1 pathogenesis.

Do We Really Need Hazard Prevention at the Expense of Safeguarding Death Dignity in COVID-19?

To date, little is known regarding the transmission risks of SARS-CoV-2 infection for subjects involved in handling, transporting, and examining deceased persons with known or suspected COVID-19 positivity at the time of death. This experimental study aims to define if and/or how long SARS-CoV-2 persists with replication capacity in the tissues of individuals who died with/from COVID-19, thereby generating infectious hazards. Sixteen patients who died with/from COVID-19 who underwent autopsy between April 2020 and April 2021 were included in this study. Based on PMI, all samples were subdivided into two groups: ‘short PMI’ group (eight subjects who were autopsied between 12 to 72 h after death); ‘long PMI’ (eight subjects who were autopsied between 24 to 78 days after death). All patients tested positive for RT-PCR at nasopharyngeal swab both before death and on samples collected during post-mortem investigation. Moreover, a lung specimen was collected and frozen at −80 °C in order to perform viral culture. The result was defined based on the cytopathic effect (subjective reading) combined with the positivity of the RT-PCR test (objective reading) in the supernatant. Only in one sample (PMI 12 h), virus vitality was demonstrated. This study, supported by a literature review, suggests that the risk of cadaveric infection in cases of a person who died from/with COVID-19 is extremely low in the first hours after death, becoming null after 12 h after death, confirming the World Health Organization (WHO) assumed in March 2020 and suggesting that the corpse of a subject who died from/with COVID-19 should be generally considered not infectious.

Mechanism-Based Cardiac Regeneration Strategies in Mammals.

Heart failure in adults is a leading cause of morbidity and mortality worldwide. It can arise from a variety of diseases, with most resulting in a loss of cardiomyocytes that cannot be replaced due to their inability to replicate, as well as to a lack of resident cardiomyocyte progenitor cells in the adult heart. Identifying and exploiting mechanisms underlying loss of developmental cardiomyocyte replicative capacity has proved to be useful in developing therapeutics to effect adult cardiac regeneration. Of course, effective regeneration of myocardium after injury requires not just expansion of cardiomyocytes, but also neovascularization to allow appropriate perfusion and resolution of injury-induced inflammation and interstitial fibrosis, but also reversal of adverse left ventricular remodeling. In addition to overcoming these challenges, a regenerative therapy needs to be safe and easily translatable. Failure to address these critical issues will delay the translation of regenerative approaches. This review critically analyzes current regenerative approaches while also providing a framework for future experimental studies aimed at enhancing success in regenerating the injured heart.

Delta variant of SARS-CoV-2: characteristics and implications for public health in Colombia

The SARS-CoV-2 Delta variant has become one of the greatest public health challenges worldwide since, after being first identified in India in December 2020, it has spread rapidly, affecting mainly countries with low vaccination rates and those that have relaxed the public health and social measures implemented to control the COVID-19 pandemic. The Delta variant has a higher replication capacity and is associated with viral loads up to 1 260 times higher than those of infections caused by the original strain, which may be associated with an increased likelihood of hospitalization, ICU admission, need for oxygen therapy, pneumonia, or even death. Fully vaccinated individuals have almost similar protection against both Delta and Alpha variants. Given the impact of Delta in countries where it is the dominant variant, it is necessary for all countries to develop systematic action plans focused on implementing strict public health and social measures in the context of the COVID-19 pandemic and on increasing vaccination coverage. Bearing this in mind, the objective of this reflection paper is to describe the main characteristics of the Delta variant, its impact on the dynamics of the pandemic in some of the countries where it has been detected, the effectiveness of vaccines against this variant, and its implications for public health in Colombia.

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.

Molecular basis for reduced cleavage activity and drug resistance in D30N HIV-1 protease

Nelfinavir is one of the FDA-approved HIV-1 protease inhibitors and a part of highly active anti-retroviral therapy (HAART) for the treatment of HIV-AIDS. Nelfinavir was the first HIV-1 protease inhibitor to be approved as a paediatric formulation. The application of HAART had resulted in significant improvement in the lives of AIDS patients. However, the emergence of drug resistance in HIV-1 protease has limited the use of many of these drugs including nelfinavir. A unique mutation observed frequently in patients treated with nelfinavir is D30N as it is selected exclusively by nelfinavir. The D30N mutation imparts very high resistance to nelfinavir but unlike other primary mutations does not give cross-resistance to the majority of other drugs. D30N mutation also significantly reduces cleavage activity of HIV-1 protease and affects viral fitness. Here, we have determined crystal structures of D30N HIV-1 protease in unliganded form and in complex with nelfinavir. These structures provide the rationale for reduced cleavage activity and the molecular basis of drug resistance induced by D30N mutation. The loss of coulombic interaction part of a crucial hydrogen bond between the drug and the protease is likely to play a major role in reduced affinity and resistance towards nelfinavir. The decreased catalytic activity of D30N HIV-1 protease due to altered interaction with the substrates and reduced stability of folding core may be the reason for the reduced replicative capacity of the virus harboring mutant HIV-1 protease. Communicated by Ramaswamy H. Sarma