Viral Replication Research Articles

Immunovirological status in people with perinatal and adult-acquired HIV-1 infection: a multi-cohort analysis from France

No study has compared the virological and immunological status of young people with perinatally-acquired HIV infection (P-HIV) with that of people with HIV adulthood (A-HIV) having a similar duration of infection. 5 French cohorts of P-HIV and A-HIV patients with a known date of HIV-infection and receiving antiretroviral treatment (ART), were used to compare the following proportions of: virological failure (VF) defined as plasma HIV RNA ≥ 50 copies/mL, CD4 cell percentages and CD4:CD8 ratios, at the time of the most recent visit since 2012. The analysis was stratified on time since infection, and multivariate models were adjusted for demographics and treatment history. 310P-HIV were compared to 1515 A-HIV (median current ages 20.9 [IQR:14.4-25.5] and 45.9 [IQR:37.9-53.5] respectively). VF at the time of the most recent evaluation was significantly higher among P-HIV (22.6%, 69/306) than A-HIV (3.3%, 50/1514); p≤0.0001. The risk of VF was particularly high among the youngest children (2-5 years), adolescents (13-17 years) and young adults (18-24 years), compared to A-HIV with a similar duration of infection: adjusted Odds-Ratio (aOR) 7.0 [95% CI: 1.7; 30.0], 11.4 [4.2; 31.2] and 3.3 [1.0; 10.8] respectively. The level of CD4 cell percentages did not differ between P-HIV and A-HIV. P-HIV aged 6-12 and 13-17 were more likely than A-HIV to have a CD4:CD8 ratio ≥ 1: 84.1% vs. 58.8% (aOR=3.5 [1.5; 8.3]), and 60.9% vs. 54.7% (aOR=1.9 [0.9; 4.2]) respectively. P-HIV were at a higher risk of VF than A-HIV with a similar duration of infection, even after adjusting for treatment history, whereas they were not at a higher risk of immunological impairment. Exposure to viral replication among young patients living with HIV since birth or a very early age, probably because of lower adherence, could have an impact on health, raising major concerns about the selection of resistance mutations and the risk of HIV transmission. Inserm - ANRS MIE.

Co-Transcriptional Regulation of HBV Replication: RNA Quality Also Matters

Chronic hepatitis B (CHB) virus infection is a major public health burden and the leading cause of hepatocellular carcinoma. Despite the efficacy of current treatments, hepatitis B virus (HBV) cannot be fully eradicated due to the persistence of its minichromosome, or covalently closed circular DNA (cccDNA). The HBV community is investing large human and financial resources to develop new therapeutic strategies that either silence or ideally degrade cccDNA, to cure HBV completely or functionally. cccDNA transcription is considered to be the key step for HBV replication. Transcription not only influences the levels of viral RNA produced, but also directly impacts their quality, generating multiple variants. Growing evidence advocates for the role of the co-transcriptional regulation of HBV RNAs during CHB and viral replication, paving the way for the development of novel therapies targeting these processes. This review focuses on the mechanisms controlling the different co-transcriptional processes that HBV RNAs undergo, and their contribution to both viral replication and HBV-induced liver pathogenesis.

Phlorizin Limits Bovine Viral Diarrhea Virus Infection in Mice via Regulating Gut Microbiota Composition.

Phlorizin (PHZ) is one of the main pharmacologically active ingredients in Lithocarpus polystachyus. We have previously shown that PHZ inhibits the replication of bovine viral diarrhea virus (BVDV), but the exact antiviral mechanism, especially in vivo, is still unknown. Here, we further confirm that PHZ has good protective effects in BVDV-infected mice. We analyzed BVDV-induced CD3+, CD4+, and CD8+ T cells among peripheral blood lymphocytes and found that PHZ significantly restored their percentage. Metagenomic analyses revealed that PHZ markedly improved the richness and diversity of intestinal microbiota and increased the abundance of potentially health-related microbes (families Lachnosipiraceae, Ruminococcaceae, and Oscillospiraceae). Specifically, the relative abundance of short chain fatty acid (SCFA)-producing bacteria, including Lachnospiraceae_UCG-006, unclassified_f_Ruminococcaceae, Oscillibacter, Intestinimonas, Blautia, and Lachnoclostridium increased significantly after PHZ treatment. Interestingly, BVDV-infected mice that received fecal microbiota from PHZ-treated mice (PHZ-FMT) had a significantly lower viral load in the duodenum and jejunum than untreated mice. Pathological lesions of duodenum and jejunum were also greatly reduced in the PHZ-FMT group, confirming a significant antiviral effect. These findings show that gut microbiota play an important role in PHZ's antiviral activity and suggest that their targeted intervention might be a promising endogenous strategy to prevent and control BVDV.

A retrospective cohort study of Paxlovid efficacy depending on treatment time in hospitalized COVID-19 patients.

Paxlovid, a SARS-CoV-2 antiviral, not only prevents severe illness but also curtails viral shedding, lowering transmission risks from treated patients. By fitting a mathematical model of within-host Omicron viral dynamics to electronic health records data from 208 hospitalized patients in Hong Kong, we estimate that Paxlovid can inhibit over 90% of viral replication. However, its effectiveness critically depends on the timing of treatment. If treatment is initiated three days after symptoms first appear, we estimate a 17% chance of a post-treatment viral rebound and a 12% (95% CI: 0%-16%) reduction in overall infectiousness for non-rebound cases. Earlier treatment significantly elevates the risk of rebound without further reducing infectiousness, whereas starting beyond five days reduces its efficacy in curbing peak viral shedding. Among the 104 patients who received Paxlovid, 62% began treatment within an optimal three-to-five-day day window after symptoms appeared. Our findings indicate that broader global access to Paxlovid, coupled with appropriately timed treatment, can mitigate the severity and transmission of SARS-Cov-2.

Design, synthesis and biological evaluation of novel 3C-like protease inhibitors as lead compounds against SARS-CoV-2.

Background: The epidemic caused by SARS-CoV-2swept the world in 2019. The 3C-like protease (3CLpro) of SARS-CoV-2 plays a key role in viral replication, and its inhibition could inhibit viral replication. Materials & methods: The virtual screen based on receptor-ligand pharmacophore models and molecular docking were conducted to obtain the novel scaffolds of the 3CLpro. The molecular dynamics simulation was also carried out. All compounds were synthesized and evaluated in biochemical assays. Results: The compound C2 could inhibit 3CLpro with a 72% inhibitory rate at 10μM. The covalent docking showed that C2 could form a covalent bond with the Cys145 in 3CLpro. Conclusion: C2 could be a potent lead compound of 3CLpro inhibitors against SARS-CoV-2.

Age-specific nasal epithelial responses to SARS-CoV-2 infection.

Children infected with SARS-CoV-2 rarely progress to respiratory failure. However, the risk of mortality in infected people over 85 years of age remains high. Here we investigate differences in the cellular landscape and function of paediatric (<12 years), adult (30-50 years) and older adult (>70 years) ex vivo cultured nasal epithelial cells in response to infection with SARS-CoV-2. We show that cell tropism of SARS-CoV-2, and expression of ACE2 and TMPRSS2 in nasal epithelial cell subtypes, differ between age groups. While ciliated cells are viral replication centres across all age groups, a distinct goblet inflammatory subtype emerges in infected paediatric cultures and shows high expression of interferon-stimulated genes and incomplete viral replication. In contrast, older adult cultures infected with SARS-CoV-2 show a proportional increase in basaloid-like cells, which facilitate viral spread and are associated with altered epithelial repair pathways. We confirm age-specific induction of these cell types by integrating data from in vivo COVID-19 studies and validate that our in vitro model recapitulates early epithelial responses to SARS-CoV-2 infection.

Molecular modelling studies and in vitro enzymatic assays identified A 4-(nitrobenzyl)guanidine derivative as inhibitor of SARS-CoV-2 Mpro

Scientists and researchers have been searching for drugs targeting the main protease (Mpro) of SARS-CoV-2, which is crucial for virus replication. This study employed a virtual screening based on molecular docking to identify benzoylguanidines from an in-house chemical library that can inhibit Mpro on the active site and three allosteric sites. Molecular docking was performed on the LaSMMed Chemical Library using 88 benzoylguanidine compounds. Based on their RMSD values and conserved pose, three potential inhibitors (BZG1, BZG2, and BZG3) were selected. These results indicate that BZG1 and BZG3 may bind to the active site, while BZG2 may bind to allosteric sites. Molecular dynamics data suggest that BZG2 selectively targets allosteric site 3. In vitro tests were performed to measure the proteolytic activity of rMpro. The tests showed that BZG2 has uncompetitive inhibitory activity, with an IC50 value of 77 µM. These findings suggest that benzoylguanidines possess potential as Mpro inhibitors and pave the way towards combating SARS-Cov-2 effectively.

Interaction and Antiviral Treatment of Coinfection between SARS-CoV-2 and Influenza in Vitro

BackgroundThe pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has lasted for three years. Coinfection with seasonal influenza may occur resulting in more severe diseases. The interaction between these two viruses for infection and the effect of antiviral treatment remains unclear. MethodsA SARS-CoV-2 and influenza H1N1 coinfection model on Calu-3 cell line was established, upon which the simultaneous and sequential coinfection was evaluated by comparing the viral load. The efficacy of molnupiravir and baloxavir against individual virus and coinfection were also studied. ResultsThe replication of SARS-CoV-2 was significantly interfered when the influenza virus was infected simultaneously or in advance (p<0.05). On the contrary, the replication of the influenza virus was not affected by the SARS-CoV-2. Molnupiravir monotherapy had significant inhibitory effect on SARS-CoV-2 when the concentration reached to 6.25μM but did not show any significant anti-influenza activity. Baloxavir was effective against influenza within the dosage range and showed significant effect of anti-SARS-CoV-2 at 16μM. In the treatment of coinfection, molnupiravir had significant effect for SARS-CoV-2 from 6.25μM to 100μM and inhibited H1N1 at 100μM (p<0.05). The tested dosage range of baloxavir can inhibit H1N1 significantly (p<0.05), while at the highest concentration of baloxavir did not further inhibit SARS-CoV-2, and the replication of SARS-CoV-2 significantly increased in lower concentrations. Combination treatment can effectively inhibit influenza H1N1 and SARS-CoV-2 replication during coinfection. Compared with molnupiravir or baloxavir monotherapy, combination therapy was more effective in less dosage to inhibit the replication of both viruses. ConclusionsIn coinfection, the replication of SARS-CoV-2 would be interfered by influenza H1N1. Compared with molnupiravir or baloxavir monotherapy, treatment with a combination of molnupiravir and baloxavir should be considered for early treatment in patients with SARS-CoV-2 and influenza coinfection.

M6A Methylation in Regulation of Antiviral Innate Immunity

The epitranscriptomic modification m6A is a prevalent RNA modification that plays a crucial role in the regulation of various aspects of RNA metabolism. It has been found to be involved in a wide range of physiological processes and disease states. Of particular interest is the role of m6A machinery and modifications in viral infections, serving as an evolutionary marker for distinguishing between self and non-self entities. In this review article, we present a comprehensive overview of the epitranscriptomic modification m6A and its implications for the interplay between viruses and their host, focusing on immune responses and viral replication. We outline future research directions that highlight the role of m6A in viral nucleic acid recognition, initiation of antiviral immune responses, and modulation of antiviral signaling pathways. Additionally, we discuss the potential of m6A as a prognostic biomarker and a target for therapeutic interventions in viral infections.

Epidemiology and Dynamics of BK Polyomavirus Replication after Kidney Transplantation

Background/Objectives: In the absence of an effective antiviral treatment for BK polyomavirus (BKPyV), a better understanding of the epidemiology and time course of BKPyV replication after kidney transplantation is needed to limit the virus’s impact on the graft outcome. Methods: In a 7-year study, we screened more than 430 kidney transplant recipients and analyzed the time course and virological characteristics of BKPyV replication. Results: Urinary viral replication was observed in 116 (27%) of the 430 patients, and 90 of the 116 (78%) had viral DNAemia. Thirty-eight patients (8.8%) were presumed to have nephropathy (DNAemia &gt; 4 log10 copies/mL). Of the patients with BKPyV replication, 48%, 60%, 71%, and 80% were first found to be positive one, two, three, and four months post-transplantation. The initial viral load in the urine was below 7 log10 copies/mL in 100% of the patients with viral replication first detected before the first month, and this proportion was 57% when viral replication was first detected after the first month. When the BKPyV replication was first detected in a urine sample at month 3 or later, 81.5% of patients had concomitant BKPyV DNAemia. The predominant viral subtype was Ib2 (60%), and there was no apparent relationship between the subtype and the time course of BKPyV replication. Conclusions: Urinary BKPyV replication occurs early after renal transplantation and in most patients will increase to a level requiring therapeutic intervention. Close monitoring for BKPyV in the early post-transplantation period would enable the pre-emptive adjustment of the immunosuppression regimen.

Mpox: An Overview of Pathogenesis, Diagnosis, and Public Health Implications

Mpox, caused by viruses of the genus Orthopoxvirus, is an emerging threat to human and animal health. With increasing urbanization and more frequent interaction between humans and wild animals, the risk of Mpox transmission to humans has increased significantly. This review aims to examine in depth the epidemiology, pathogenesis, and diagnosis of Mpox, with a special focus on recent discoveries and advances in understanding the disease. Molecular mechanisms involved in viral replication will be examined, as well as risk factors associated with interspecific transmission and spread of the disease in human populations. Currently available diagnostic methods will also be discussed, with a critical analysis of their limitations and possible future directions for improving the accuracy and timeliness of diagnosis. Finally, this review will explore the public health implications associated with Mpox, emphasizing the importance of epidemiological surveillance, vaccination, and emergency preparedness to prevent and manage possible outbreaks. Understanding the epidemiology and control strategies for Mpox is critical to protecting the health of human and animal communities and mitigating the risk of interspecific transmission and spread of the disease.

Phylogenetic analysis of the promoter element 2 of paramyxo- and filoviruses.

The genomic intricacies of paramyxo- and filoviruses are highlighted by the bipartite promoters-promoter element 1 (PE1) and promoter element 2 (PE2)-at their 3' termini. The spacer region between these elements follows the "rule of six," crucial for genome replication. By a comprehensive analysis of paramyxoviral genome sequences, we identified distinct subcategories of PE2 based on C and CG repeats that were specific to Orthoparamyxovirinae and Avulavirinae/Rubulavirinae, respectively, mirroring their evolutionary lineages. Notably, the PE2 of Rubulavirinae is integrated into the gene-coding region, a unique trait potentially linked to its strict dependence on RNA editing for virus growth. This study also focused on the PE2 sequences in filovirus genomes. The strict conservation of the continuity of UN5 among virus species emphasizes its crucial role in viral genome replication.

Nonnegligible Contribution of Nonlymphoid Tissue to Viral Reservoir During the Short-Term Early cART in SIVmac239-Infected Chinese Rhesus Macaques.

HIV/AIDS cannot be cured because of the persistence of the viral reservoir. Because of the complexity of the cellular composition and structure of the human organs, HIV reservoirs of anatomical site are also complex. Recently, although a variety of molecules have been reported to be involved in the establishment and maintenance of the viral reservoirs, or as marker of latent cells, the research mainly focuses on blood and lymph nodes. Now, the characteristics of the viral reservoir in tissue are not yet fully understood. In this study, various tissues were collected from SIVmac239-infected monkeys, and the level of total SIV DNA, SIV 2-LTR DNA, and cell-associated virus RNA in them were compared with character of the anatomical viral reservoir under early treatment. The results showed that short-term combination antiretroviral therapy (cART) starting from 3 days after infection could significantly inhibit viremia and reduce the size of the anatomical viral reservoir, but it could not eradicate de novo infections and ongoing replication of virus. Moreover, the effects of early cART on the level of total SIV DNA, SIV 2-LTR DNA, and cell-associated virus RNA in different tissues were different, which changed the size distribution of viral reservoir in anatomical site. Finally, the contribution of nonlymphoid tissues, especially liver and lung, to the viral reservoir increased after treatment, while the contribution of intestinal lymphoid to the viral reservoir significantly reduced. These results suggested that early treatment effectively decreased the size of viral reservoir, and that the effects of cART on the tissue viral reservoir varied greatly by tissue type. The results implied that persistent existence of virus in nonlymphoid tissues after short-term treatment suggested that the role of nonlymphoid tissues cannot be ignored in development strategies for AIDS therapy.

Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease.

We have witnessed three coronavirus (CoV) outbreaks in the past two decades, including the COVID-19 pandemic caused by SARS-CoV-2. Main protease (MPro), a highly conserved protease among various CoVs, is essential for viral replication and pathogenesis, making it a prime target for antiviral drug development. Here, we leverage proteolysis targeting chimera (PROTAC) technology to develop a new class of small-molecule antivirals that induce the degradation of SARS-CoV-2 MPro. Among them, MPD2 was demonstrated to effectively reduce MPro protein levels in 293T cells, relying on a time-dependent, CRBN-mediated, and proteasome-driven mechanism. Furthermore, MPD2 exhibited remarkable efficacy in diminishing MPro protein levels in SARS-CoV-2-infected A549-ACE2 cells. MPD2 also displayed potent antiviral activity against various SARS-CoV-2 strains and exhibited enhanced potency against nirmatrelvir-resistant viruses. Overall, this proof-of-concept study highlights the potential of targeted protein degradation of MPro as an innovative approach for developing antivirals that could fight against drug-resistant viral variants.

Porcine reproductive and respiratory syndrome virus 2 hijacks CMA-mediated lipolysis through upregulation of small GTPase RAB18.

RAB GTPases (RABs) control intracellular membrane trafficking with high precision. In the present study, we carried out a short hairpin RNA (shRNA) screen focused on a library of 62 RABs during infection with porcine reproductive and respiratory syndrome virus 2 (PRRSV-2), a member of the family Arteriviridae. We found that 13 RABs negatively affect the yield of PRRSV-2 progeny virus, whereas 29 RABs have a positive impact on the yield of PRRSV-2 progeny virus. Further analysis revealed that PRRSV-2 infection transcriptionally regulated RAB18 through RIG-I/MAVS-mediated canonical NF-κB activation. Disrupting RAB18 expression led to the accumulation of lipid droplets (LDs), impaired LDs catabolism, and flawed viral replication and assembly. We also discovered that PRRSV-2 co-opts chaperone-mediated autophagy (CMA) for lipolysis via RAB18, as indicated by the enhanced associations between RAB18 and perlipin 2 (PLIN2), CMA-specific lysosomal associated membrane protein 2A (LAMP2A), and heat shock protein family A (Hsp70) member 8 (HSPA8/HSC70) during PRRSV-2 infection. Knockdown of HSPA8 and LAMP2A impacted on the yield of PRRSV-2 progeny virus, implying that the virus utilizes RAB18 to promote CMA-mediated lipolysis. Importantly, we determined that the C-terminal domain (CTD) of HSPA8 could bind to the switch II domain of RAB18, and the CTD of PLIN2 was capable of associating with HSPA8, suggesting that HSPA8 facilitates the interaction between RAB18 and PLIN2 in the CMA process. In summary, our findings elucidate how PRRSV-2 hijacks CMA-mediated lipid metabolism through innate immune activation to enhance the yield of progeny virus, offering novel insights for the development of anti-PRRSV-2 treatments.

Characterizing changes in transcriptome and kinome responses in testicular cells during infection by Ebola virus

Ebola virus (EBOV) is able to persist and actively replicate in the reproductive tract of male disease survivors months or years after recovery from Ebola virus disease (EVD)1. Persistent EBOV infections are usually asymptomatic and can be transmitted sexually, but the host and viral factors that mediate these infections have not been characterized2,3. We investigated the interaction between host and viral factors during EBOV infection of the blood testis barrier (BTB), with a focus on Sertoli cells as a potential reservoir for viral persistence. We assessed viral replication kinetics and host responses of mouse testicular Leydig cells and Sertoli cells infected with EBOV Makona (i.e. infectious EBOV) and collected samples up to 28 days post-infection. Viral replication was apparent in both cell lines, but intracellular early viral loads were much higher in Leydig cells compared to Sertoli cells. We used RNAseq analysis to characterize transcriptomic responses of Leydig cells and Sertoli cells to EBOV infection over time. Further investigation of early interactions between host cells and EBOV was performed using virus-like particles (EBOV trVLP) and assays of phosphorylation-based cell signaling. Our findings indicate that virus-treated Sertoli cells responded more rapidly and robustly than Leydig cells, and with a particular emphasis on detection of, and response to, external stimuli. We discuss how the roles played by Sertoli cells in immune privilege and spermatogenesis may affect their initial and continued response to EBOV infection in a manner that could facilitate asymptomatic persistence.

Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As.

Although the significance of chemical modifications on RNA is acknowledged, the evolutionary benefits and specific roles in human immunodeficiency virus (HIV-1) replication remain elusive. Most studies have provided only population-averaged values of modifications for fragmented RNAs at low resolution and have relied on indirect analyses of phenotypic effects by perturbing host effectors. Here we analysed chemical modifications on HIV-1 RNAs at the full-length, single RNA level and nucleotide resolution using direct RNA sequencing methods. Our data reveal an unexpectedly simple HIV-1 modification landscape, highlighting three predominant N6-methyladenosine (m6A) modifications near the 3' end. More densely installed in spliced viral messenger RNAs than in genomic RNAs, these m6As play a crucial role in maintaining normal levels of HIV-1 RNA splicing and translation. HIV-1 generates diverse RNA subspecies with distinct m6A ensembles, and maintaining multiple of these m6As on its RNAs provides additional stability and resilience to HIV-1 replication, suggesting an unexplored viral RNA-level evolutionary strategy.

Distinct domain organization and diversity of 2'-5'-oligoadenylate synthetases.

The 2'-5' oligoadenylate synthetases (OAS) are important components of the innate immune system that recognize viral double stranded RNA (dsRNA). Upon dsRNA binding, OAS generate 2'-5'-linked oligoadenylates (2-5A) that activate ribonuclease L (RNase L), halting viral replication. The OAS/RNase L pathway is thus an important antiviral pathway and viruses have devised strategies to circumvent OAS activation. OAS enzymes are divided into four classes according to size: small (OAS1), medium (OAS2) and large (OAS3) that consists of one, two and three OAS domains respectively, and the OAS-like protein (OASL) that consists of one OAS domain and tandem domains similar to ubiquitin. Early investigation of the OAS enzymes hinted at the recognition of dsRNA by OAS, but due to size differences amongst OAS family members combined with the lack of structural information on full-length OAS2 and OAS3, the regulation of OAS catalytic activity by dsRNA was not well understood. However, the recent biophysical studies of OAS have highlighted overall structure and domain organization. In this review, we present a detailed examination of the OAS literature and summarized the investigation on 2' 5'-oligoadenylate synthetases.

Special Issue “Viral-Induced Inflammation”

Inflammation is a protective host response essential for controlling viral replication and promoting tissue repair [...]

The W195 Residue of the Newcastle Disease Virus V Protein Is Critical for Multiple Aspects of Viral Self-Regulation through Interactions between V and Nucleoproteins

The transcription and replication of the Newcastle disease virus (NDV) strictly rely on the viral ribonucleoprotein (RNP) complex, which is composed of viral NP, P, L and RNA. However, it is not known whether other viral non-RNP proteins participate in this process for viral self-regulation. In this study, we used a minigenome (MG) system to identify the regulatory role of the viral non-RNP proteins V, M, W, F and HN. Among them, V significantly reduced MG-encoded reporter activity compared with the other proteins and inhibited the synthesis of viral mRNA and cRNA. Further, V interacted with NP. A mutation in residue W195 of V diminished V–NP interaction and inhibited inclusion body (IB) formation in NP-P-L-cotransfected cells. Furthermore, a reverse-genetics system for the highly virulent strain F48E9 was established. The mutant rF48E9-VW195R increased viral replication and apparently enhanced IB formation. In vivo experiments demonstrated that rF48E9-VW195R decreased virulence and retarded time of death. Overall, the results indicate that the V–NP interaction of the W195 mutant V decreased, which regulated viral RNA synthesis, IB formation, viral replication and pathogenicity. This study provides insight into the self-regulation of non-RNP proteins in paramyxoviruses.