Virus Replication In Cell Culture Research Articles

Detection of the first recombinant African swine fever virus (genotypes I and II) in domestic pigs in Russia.

African swine fever (ASF) is a contagious viral disease that affects pigs and wild boars, with a mortality rate of up to 100% in susceptible animals. The virus has been circulating in Europe and Asia since its introduction in 2007. Initially, all studied isolates were identified as genotype II, but in 2021 genotype I was reported in China. Later in 2023, the first recombinant virus of genotype I and II was identified in China, with an isolate dating back to 2021, this was followed by the detection of 6 recombinant isolates in Vietnam. In this study, an ASFV isolate from the Primorsky Region of Russia obtained from a domestic pig was analyzed by sequencing several genome markers as well as the full genome. Eight pigs were infected with the isolate to assess its virulence. Virus replication in cell culture showed hemadsorption, while sequencing of genome markers clustered the isolate into both genotype I and genotype II. The whole-genome sequence showed that the Russian isolate shared a 99.99% identity with recombinant isolates described earlier in China. Experimental animals developed ASF disease after the introduction of a low dose of the virus (10 HAU50) and died within 7 days post-infection, presenting an acute form of the disease. This is the first report on recombinant ASFV in Russia's territory. The results once again confirm the transboundary nature of the disease, demonstrating the vulnerability of the global pig industry underscoring the need for developing new ASF vaccines effective against recombinant strains and emphasizing the importance of continuous molecular monitoring to detect emerging threats promptly.

The dual role of a highly structured RNA (the S fragment) in the replication of foot-and-mouth disease virus.

Secondary and tertiary RNA structures play key roles in genome replication of single-stranded positive sense RNA viruses. Complex, functional structures are particularly abundant in the untranslated regions of picornaviruses, where they are involved in initiation of translation, priming of new strand synthesis and genome circularization. The 5' UTR of foot-and-mouth disease virus (FMDV) is predicted to include a c. 360 nucleotide-long stem-loop, termed the short (S) fragment. This structure is highly conserved and essential for viral replication, but the precise function(s) are unclear. Here, we used selective 2' hydroxyl acetylation analyzed by primer extension (SHAPE) to experimentally determine aspects of the structure, alongside comparative genomic analyses to confirm structure conservation from a wide range of field isolates. To examine its role in virus replication in cell culture, we introduced a series of deletions to the distal and proximal regions of the stem-loop. These truncations affected genome replication in a size-dependent and, in some cases, host cell-dependent manner. Furthermore, during the passage of viruses incorporating the largest tolerated deletion from the proximal region of the S fragment stem-loop, an additional mutation was selected in the viral RNA-dependent RNA polymerase, 3Dpol. These data suggest that the S fragment and 3Dpol interact in the formation of the FMDV replication complex.

Non-Analog Compounds to Sialic Acid as Inhibitors of Influenza Virus Neuraminidase: An Underexplored Approach for Novel Antivirals―Systematic Review

Influenza poses a significant threat to public health worldwide, particularly among vulnerable populations such as children, the elderly, immunocompromised individuals, and those with chronic diseases. It is associated with high mortality and morbidity rates. Neuraminidase inhibitors play a crucial role in influenza treatment by mitigating the risk of complications and death. However, the genetic variability of the influenza virus enables the emergence of drug-resistant mutations. This review focuses on the search for new compounds that are not analogous to sialic acid, aiming to inhibit the activity of viral neuraminidase in vitro, viral replication in cell cultures, or animal models. Influenza virus strains that have been reported in the literature present specific mutations that generate resistance to neuraminidase inhibitors. Since these inhibitors bear structural resemblance to sialic acid, the predominant location for these mutations is the enzyme’s active site. Consequently, exploring alternative compound classes becomes imperative to circumvent this interaction pattern. These compounds will introduce diverse molecular frameworks, serving as foundational structures for further development through rational drug design, thereby engendering novel antiviral agents targeting influenza. The potential prospects for developing novel influenza antivirals based on these findings are discussed.

CX-6258 hydrochloride hydrate: A potential non-nucleoside inhibitor targeting the RNA-dependent RNA polymerase of norovirus

Human norovirus (HuNoV), a primary cause of non-bacterial gastroenteritis, currently lacks approved treatment. RdRp is vital for virus replication, making it an attractive target for therapeutic intervention. By application of structure-based virtual screening procedure, we present CX-6258 hydrochloride hydrate as a potent RdRp non-nucleoside inhibitor, effectively inhibiting HuNoV RdRp activity with an IC50 of 3.61 μM. Importantly, this compound inhibits viral replication in cell culture, with an EC50 of 0.88 μM. In vitro binding assay validate that CX-6258 hydrochloride hydrate binds to RdRp through interaction with the “B-site” binding pocket. Interestingly, CX-6258-contacting residues such as R392, Q439, and Q414 are highly conserved among major norovirus GI and GII variants, suggesting that it may be a general inhibitor of norovirus RdRp. Given that CX-6258 hydrochloride hydrate is already utilized as an orally efficacious pan-Pim kinase inhibitor, it may serve as a potential lead compound in the effort to control HuNoV infections.

SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in cell culture and in mice

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in these drug targets is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein encoded as a small domain at the N terminus of nonstructural protein 3. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and IFN-stimulated gene expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.

HIV-1 Integrase Inhibition Activity by Spiroketals Derived from Plagius flosculosus, an Endemic Plant of Sardinia (Italy) and Corsica (France).

In this work we investigated, for the first time, the effect of Plagius flosculosus (L.) Alavi & Heywood, a Sardinian-Corsican endemic plant, on HIV-1 integrase (IN) activity. The phytochemical analysis of the leaves chloroform extract led us to isolate and characterize three compounds (SPK1, SPK2, and SPK3) belonging to the spiroketals, a group of naturally occurring metabolites of phytochemical relevance with interesting biological properties. Due to their structural diversity, these cyclic ketals have attracted the interest of chemists and biologists. SPK1, SPK2, and SPK3 were evaluated here for their ability to inhibit HIV-1 integrase activity in biochemical assays. The results showed that all the compounds inhibited HIV-1 IN activity. In particular, the most active one was SPK3, which interfered in a low molecular range (IC50 of 1.46 ± 0.16 µM) with HIV-1 IN activity in the presence/absence of the LEDGF cellular cofactor. To investigate the mechanism of action, the three spiroketals were also tested on HIV-1 RT-associated Ribonuclease H (RNase H) activity, proving to be active in inhibiting this function. Although SPK3 was unable to inhibit viral replication in cell culture, it promoted the IN multimerization. We hypothesize that SPK3 inhibited HIV-1 IN through an allosteric mechanism of action.

Small but Highly Versatile: The Viral Accessory Protein Vpu.

Human and simian immunodeficiency viruses (HIVs and SIVs, respectively) encode several small proteins (Vif, Vpr, Nef, Vpu, and Vpx) that are called accessory because they are not generally required for viral replication in cell culture. However, they play complex and important roles for viral immune evasion and spread in vivo. Here, we discuss the diverse functions and the relevance of the viral protein U (Vpu) that is expressed from a bicistronic RNA during the late stage of the viral replication cycle and found only in HIV-1 and closely related SIVs. It is well established that Vpu counteracts the restriction factor tetherin, mediates degradation of the primary viral CD4 receptors, and inhibits activation of the transcription factor nuclear factor kappa B. Recent studies identified additional activities and provided new insights into the sophisticated mechanisms by which Vpu enhances and prolongs the release of fully infectious viral particles. In addition, it has been shown that Vpu prevents superinfection not only by degrading CD4 but also by modulating DNA repair mechanisms to promote degradation of nuclear viral complementary DNA in cells that are already productively infected.

Improvement of protein production in baculovirus expression vector system by removing a total of 10 kb of nonessential fragments from Autographa californica multiple nucleopolyhedrovirus genome.

Baculovirus expression vector system (BEVS) is a powerful and versatile platform for recombinant protein production in insect cells. As the most frequently used baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes 155 open reading frames (ORFs), including a considerable number of non-essential genes for the virus replication in cell culture. Studies have shown that protein production in BEVS can be improved by removing some viral dispensable genes, and these AcMNPV vectors also offer the possibility of accommodating larger exogenous gene fragments. In this study, we, respectively, deleted 14 DNA fragments from AcMNPV genome, each of them containing at least two contiguous genes that were known nonessential for viral replication in cell culture or functionally unknown. The effects of these fragment-deletions on virus replication and exogenous protein production were examined. The results showed that 11 of the 14 fragments, containing 43 genes, were dispensable for the virus replication in cultured cells. By detecting the expression of intracellularly expressed and secreted reporter proteins, we demonstrated that nine of the fragment-deletions benefited protein production in Sf9 cells and/or in High Five cells. After combining the deletion of some dispensable fragments, we obtained two AcMNPV vectors shortened by more than 10 kb but displayed an improved capacity for recombinant protein production. The deletion strategies used in this study has the potential to further improve the BEVS.

Rift Valley fever (RVF): a re-emerging zoonotic disease, pathogenesis, epidemiology, current status, and future perspective - correspondence.

Rift Valley fever (RVF): a re-emerging zoonotic disease, pathogenesis, epidemiology, current status, and future perspective - correspondence.

A novel compound targets the feline infectious peritonitis virus nucleocapsid protein and inhibits viral replication in cell culture

Feline infectious peritonitis (FIP) is a serious viral illness in cats, caused by feline coronavirus. Once a cat develops clinical FIP, the prognosis is poor. The effective treatment strategy for coronavirus infections with immunopathological complications such as SARS-CoV-2, MERS, and FIP is focused on antiviral and immunomodulatory agents to inhibit virus replication and enhance the protective immune response. In this article we report the binding and conformational alteration of feline alphacoronavirus (FCoV) nucleocapsid protein by a novel compound K31. K31 noncompetitively inhibited the interaction between the purified nucleocapsid protein and the synthetic 5' terminus of viral genomic RNA invitro. K31 was well tolerated by cells and inhibited FCoV replication in cell culture with a selective index of 115. A single dose of K31inhibited FCoV replication to an undetectable level in 24h post treatment. K31 did not affect the virus entry to the host cell but inhibited the postentry steps of virus replication. The nucleocapsid protein forms ribonucleocapsid in association with the viral genomic RNA that serves as a template for transcription and replication of the viral genome. Our results show that K31 treatment disrupted the structural integrity of ribonucleocapsid in virus-infected cells. After the COVID-19 pandemic, most of the antiviral drug development strategies have focused on RdRp and proteases encoded by the viral genome. Our results have shown that nucleocapsid protein is a druggable target for anticoronavirus drug discovery.

Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

A fish herpesvirus highlights functional diversities among Zα domains related to phase separation induction and A-to-Z conversion.

Zalpha (Zα) domains bind to left-handed Z-DNA and Z-RNA. The Zα domain protein family includes cellular (ADAR1, ZBP1 and PKZ) and viral (vaccinia virus E3 and cyprinid herpesvirus 3 (CyHV-3) ORF112) proteins. We studied CyHV-3 ORF112, which contains an intrinsically disordered region and a Zα domain. Genome editing of CyHV-3 indicated that the expression of only the Zα domain of ORF112 was sufficient for normal viral replication in cell culture and virulence in carp. In contrast, its deletion was lethal for the virus. These observations revealed the potential of the CyHV-3 model as a unique platform to compare the exchangeability of Zα domains expressed alone in living cells. Attempts to rescue the ORF112 deletion by a broad spectrum of cellular, viral, and artificial Zα domains showed that only those expressing Z-binding activity, the capacity to induce liquid-liquid phase separation (LLPS), and A-to-Z conversion, could rescue viral replication. For the first time, this study reports the ability of some Zα domains to induce LLPS and supports the biological relevance of dsRNA A-to-Z conversion mediated by Zα domains. This study expands the functional diversity of Zα domains and stimulates new hypotheses concerning the mechanisms of action of proteins containing Zα domains.

Effectiveness of Household Disinfection Techniques to Remove SARS-CoV-2 from Cloth Masks.

To assess the efficacy of washing cloth masks, we simulated SARS-CoV-2 contamination in tricoline fabric and tested decontaminants to reduce viral particles. Viral suspensions using two variants (B.1.1.28 and P.1) were inoculated in these fabrics, and the inactivation kinetics were evaluated after washing with various household disinfection products (Soap powder, Lysoform®, Hypochlorite sodium and 70% Alcohol), rinse numbers, and exposure times. Afterward, the fabrics were washed in sterile water, and viral RNA was extracted and amplified using RT-qPCR. Finally, viral replication in cell cultures was examined. Our findings show that all biocidal treatments successfully disinfected the tissue tested. Some products showed less reduction in viral loads, such as soap powder (1.60 × 104, 1.04 × 103), soap powder and Lysoform® (1.60 × 104, 1.04 × 103), and alcohol 70% (1.02 × 103, 5.91 × 101), respectively. However, when sodium hypochlorite was used, this reduction was significantly increased (viral inactivation in 100% of the washes). After the first wash, the reduction in the number of viral particles was greater for the P.1 variant than for the B.1.1.28 variant (W = 51,759, p < 0.05). In conclusion, the role of sodium hypochlorite in cloth mask disinfection may also have implications for future health emergencies as well as recommendation by WHO.

Alphaherpesvirus US3 protein-mediated inhibition of the m6A mRNA methyltransferase complex.

SUMMARYChemical modifications of mRNA, the so-called epitranscriptome, represent an additional layer of post-transcriptional regulation of gene expression. The most common epitranscriptomic modification, N6-methyladenosine (m6A), is generated by a multi-subunit methyltransferase complex. We show that alphaherpesvirus kinases trigger phosphorylation of several components of the m6A methyltransferase complex, including METTL3, METTL14, and WTAP, which correlates with inhibition of the complex and a near complete loss of m6A levels in mRNA of virus-infected cells. Expression of the viral US3 protein is necessary and sufficient for phosphorylation and inhibition of the m6A methyltransferase complex. Although m6A methyltransferase complex inactivation is not essential for virus replication in cell culture, the consensus m6A methylation motif is under-represented in alphaherpesvirus genomes, suggesting evolutionary pressure against methylation of viral transcripts. Together, these findings reveal that phosphorylation can be associated with inactivation of the m6A methyltransferase complex, in this case mediated by the viral US3 protein.

Going Retro, Going Viral: Experiences and Lessons in Drug Discovery from COVID-19.

The severity of the COVID-19 pandemic and the pace of its global spread have motivated researchers to opt for repurposing existing drugs against SARS-CoV-2 rather than discover or develop novel ones. For reasons of speed, throughput, and cost-effectiveness, virtual screening campaigns, relying heavily on in silico docking, have dominated published reports. A particular focus as a drug target has been the principal active site (i.e., RNA synthesis) of RNA-dependent RNA polymerase (RdRp), despite the existence of a second, and also indispensable, active site in the same enzyme. Here we report the results of our experimental interrogation of several small-molecule inhibitors, including natural products proposed to be effective by in silico studies. Notably, we find that two antibiotics in clinical use, fidaxomicin and rifabutin, inhibit RNA synthesis by SARS-CoV-2 RdRp in vitro and inhibit viral replication in cell culture. However, our mutagenesis studies contradict the binding sites predicted computationally. We discuss the implications of these and other findings for computational studies predicting the binding of ligands to large and flexible protein complexes and therefore for drug discovery or repurposing efforts utilizing such studies. Finally, we suggest several improvements on such efforts ongoing against SARS-CoV-2 and future pathogens as they arise.

Evaluation of an ASFV RNA Helicase Gene A859L for Virus Replication and Swine Virulence.

African swine fever virus (ASFV) is producing a devastating pandemic that, since 2007, has spread to a contiguous geographical area from central Europe to Asia. In July 2021, ASFV was detected in the Dominican Republic, the first report of the disease in the Americas in more than 40 years. ASFV is a large, highly complex virus harboring a large dsDNA genome that encodes for more than 150 genes. The majority of these genes have not been functionally characterized. Bioinformatics analysis predicts that ASFV gene A859L encodes for an RNA helicase, although its function has not yet been experimentally assessed. Here, we evaluated the role of the A859L gene during virus replication in cell cultures and during infection in swine. For that purpose, a recombinant virus (ASFV-G-∆A859L) harboring a deletion of the A859L gene was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. Recombinant ASFV-G-∆A859L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, demonstrating that the A859L gene is non-essential for ASFV replication. Experimental infection of domestic pigs demonstrated that ASFV-G-∆A859L replicates as efficiently and induces a clinical disease indistinguishable from that caused by the parental ASFV-G. These studies conclude that the predicted RNA helicase gene A859L is not involved in the processes of virus replication or disease production in swine.

MicroRNA 122 Affects both the Initiation and the Maintenance of Hepatitis C Virus Infections.

A liver-specific microRNA, miR-122, anneals to the hepatitis C virus (HCV) genomic 5' terminus and is essential for virus replication in cell culture. However, bicistronic HCV replicons and full-length RNAs with specific mutations in the 5' untranslated region (UTR) can replicate, albeit to low levels, without miR-122. In this study, we have identified that HCV RNAs lacking the structural gene region or having encephalomyocarditis virus internal ribosomal entry site (EMCV IRES)-regulated translation had reduced requirements for miR-122. In addition, we found that a smaller proportion of cells supported miR-122-independent replication compared a population of cells supporting miR-122-dependent replication, while viral protein levels per positive cell were similar. Further, the proportion of cells supporting miR-122-independent replication increased with the amount of viral RNA delivered, suggesting that establishment of miR-122-independent replication in a cell is affected by the amount of viral RNA delivered. HCV RNAs replicating independently of miR-122 were not affected by supplementation with miR-122, suggesting that miR-122 is not essential for maintenance of an miR-122-independent HCV infection. However, miR-122 supplementation had a small positive impact on miR-122-dependent replication, suggesting a minor role in enhancing ongoing virus RNA accumulation. We suggest that miR-122 functions primarily to initiate an HCV infection but has a minor influence on its maintenance, and we present a model in which miR-122 is required for replication complex formation at the beginning of an infection and also supports new replication complex formation during ongoing infection and after infected cell division. IMPORTANCE The mechanism by which miR-122 promotes the HCV life cycle is not well understood, and a role in directly promoting genome amplification is still debated. In this study, we have shown that miR-122 increases the rate of viral RNA accumulation and promotes the establishment of an HCV infection in a greater number of cells than in the absence of miR-122. However, we also confirm a minor role in promoting ongoing virus replication and propose a role in the initiation of new replication complexes throughout a virus infection. This study has implications for the use of anti-miR-122 as a potential HCV therapy.

An all-out assault on SARS-CoV-2 replication.

The coronavirus pandemic has had a huge impact on public health with over 165 million people infected, 3.4 million deaths and a hugely deleterious effect on most economies. While vaccination effectively protects against the disease it is likely that viruses will evolve that can replicate in hosts immunised with the present vaccines. Thus, there is a great unmet need for effective antivirals that can block the development of serious disease in infected patients. The seven papers published in this issue of the Biochemical Journal address this need by expressing and purifying components required for viral replication, developing biochemical assays for these components and using the assays to screen a library of pre-existing pharmaceuticals for drugs that inhibited the target in vitro and inhibited viral replication in cell culture. The candidate drugs obtained are potential antivirals that may protect against SARS-CoV-2 infection. While not all the antiviral candidates will make it through to the clinic, they will be useful tool compounds and can act as the starting point for further drug discovery programmes.

Fe-S cofactors in the SARS-CoV-2 RNA-dependent RNA polymerase are potential antiviral targets

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.

Hepatitis C virus drugs that inhibit SARS-CoV-2 papain-like protease synergize with remdesivir to suppress viral replication in cell culture.

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2. We assessed 10 hepatitis C virus (HCV) protease-inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 main protease (Mpro) and HCV NS3/4A protease. Virtual docking experiments show that these HCV drugs can potentially bind into the Mpro substrate-binding cleft. We show that seven HCV drugs inhibit both SARS-CoV-2 Mpro protease activity and SARS-CoV-2 virus replication in Vero and/or human cells. However, their Mpro inhibiting activities did not correlate with their antiviral activities. This conundrum is resolved by demonstrating that four HCV protease inhibitor drugs, simeprevir, vaniprevir, paritaprevir, and grazoprevir inhibit the SARS CoV-2 papain-like protease (PLpro). HCV drugs that inhibit PLpro synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir’s antiviral activity as much as 10-fold, while those that only inhibit Mpro do not synergize with remdesivir.