Viral Polymerase Research Articles

Personalized Assessment of Mortality Risk and Hospital Stay Duration in Hospitalized Patients with COVID-19 Treated with Remdesivir: A Machine Learning Approach.

Background: Despite advancements in vaccination, early treatments, and understanding of SARS-CoV-2, its impact remains significant worldwide. Many patients require intensive care due to severe COVID-19. Remdesivir, a key treatment option among viral RNA polymerase inhibitors, lacks comprehensive studies on factors associated with its effectiveness. Methods: We conducted a retrospective study in 2022, analyzing data from 252 hospitalized COVID-19 patients treated with remdesivir. Six machine learning algorithms were compared to predict factors influencing remdesivir's clinical benefits regarding mortality and hospital stay. Results: The extreme gradient boost (XGB) method showed the highest accuracy for both mortality (95.45%) and hospital stay (94.24%). Factors associated with worse outcomes in terms of mortality included limitations in life support, ventilatory support needs, lymphopenia, low albumin and hemoglobin levels, flu and/or coinfection, and cough. For hospital stay, factors included vaccine doses, lung density, pulmonary radiological status, comorbidities, oxygen therapy, troponin, lactate dehydrogenase levels, and asthenia. Conclusions: These findings underscore XGB's effectiveness in accurately categorizing COVID-19 patients undergoing remdesivir treatment.

A Potential Nervous Necrosis Virus (NNV) Live Vaccine for Sole Obtained by Genomic Modification.

Viral Encephalopathy and Retinopathy (VER) is a neurological infectious fish disease that causes vacuolization and necrosis in the central nervous system, which lead to swimming abnormalities and, generally, host death in the early stages of development. VER is caused by the Nervous Necrosis Virus (NNV), a non-enveloped virus with a bisegmented and positive-stranded (+) RNA genome. The largest segment (RNA1) codes for viral polymerase while capsid protein is encoded by RNA2. The aim of this study was to explore the potential of a reverse-engineered RGNNV/SJNNV strain that harbors mutations in both 3'NCRs (position 3073 of RNA1 and 1408 and 1412 of RNA2) as an attenuated live vaccine for sole. The attenuation of this strain was confirmed through experimental infections in sole at 22 °C. Vaccination trials were performed by bath, intramuscular, and intraperitoneal injection, at two temperatures (18 and 22 °C). Our results indicate the improved survival of vaccinated fish and delayed and poorer viral replication, as well as an overexpression of immune response genes linked to T cell markers (cd4 and cd8), to an early inflammatory response (tlr7 and tnfα), and to antiviral activity (rtp3 and mx). In conclusion, our study indicates that the attenuated strain is a good vaccine candidate as it favors sole survival upon infection with the wt strain while inducing a significant immune response.

Is SARS-CoV-2 Now More Like the Seasonal Coronaviruses Following Its Evolution?

SARS-CoV-2 has evolved significantly since the beginning of the COVID-19 pandemic. COVID-19 mortality has decreased due to increased population immunity and possibly the reduced intrinsic severity of the new variants. SARS-CoV-2 is now considered an endemic virus, but the extent to which its clinical findings resemble those of seasonal coronaviruses (sCoV) is not fully understood. Pediatric patients under 18 years of age who were sent for SARS-CoV-2 polymerase chain reaction real-time reverse transcription polymerase chain reaction between January 1, 2022 and January 14, 2023 and whose results were positive were included in this study. To include only symptomatic COVID-19 patients in the study, asymptomatic patients who were positive in their screening tests were excluded. For the sCoV patients' group, patients who had a respiratory viral polymerase chain reaction assay between January 10, 2022 and January 11, 2023 and were positive for any type of sCoV were included in the study. The 2 groups were compared for clinical and laboratory characteristics. The study included 213 patients with COVID-19 and 194 patients with sCoV infection. Fever was a more common symptom in patients with COVID-19. sCoV was associated with lower respiratory involvement while increasing age was protective. The likelihood of hospitalization was decreased by increasing age but increased by the presence of comorbid conditions and lower respiratory tract involvement. The type of virus had no effect on the likelihood of hospitalization. In conclusion, sCoV infections carry a higher risk for lower respiratory involvement than COVID-19, and COVID-19 has a milder course than sCoV infections in children.

Treatment with Remdesivir of Children with SARS-CoV-2 Infection: Experience from a Clinical Hospital in Romania.

The COVID-19 pandemic was characterized by mild-to-moderate disease in children and adolescents, with low incidences of severe cases and mortality. Most of the information on drug therapy in COVID-19-positive children was derived from research in adult patients. Remdesivir, an inhibitor of viral RNA polymerase, was shown to be effective in COVID-19 patients with moderate-to-severe disease. In this study, we present our experience of the use of remdesivir in pediatric patients hospitalized with COVID-19. This retrospective study was based on the early use of remdesivir in 14 children with mild, moderate, and severe clinical forms of COVID-19, who were hospitalized between 1 January 2022, and 30 September 2023. The patients included eight infants and six children older than 1 day (the age range was 2 months to 17 years). Most of them (92.85%) had documented pneumonia. Four patients had associated acute laryngitis, and another had bronchiolitis. Coinfections with Streptococcus pneumoniae were diagnosed in two patients. The clinical course was favorable in 12/14 (85.71%) children. Two patients were transferred to the pediatric intensive care unit because of aggravation of associated acute diseases (acute laryngitis and bronchiolitis, respectively). Mild increases in alanine aminotransferase levels occurred in two patients, with no increase in serum creatinine, during treatment with remdesivir. The appropriate use of remdesivir proved safe and efficient in our group of patients. However, further studies are required to support the efficiency, tolerability, and safety of remdesivir in children.

Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories

The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication.

BAG6 inhibits influenza A virus replication by inducing viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly.

The interaction between influenza A virus (IAV) and host proteins is an important process that greatly influences viral replication and pathogenicity. PB2 protein is a subunit of viral ribonucleoprotein (vRNP) complex playing distinct roles in viral transcription and replication. BAG6 (BCL2-associated athanogene 6) as a multifunctional host protein participates in physiological and pathological processes. Here, we identify BAG6 as a new restriction factor for IAV replication through targeting PB2. For both avian and human influenza viruses, overexpression of BAG6 reduced viral protein expression and virus titers, whereas deletion of BAG6 significantly enhanced virus replication. Moreover, BAG6-knockdown mice developed more severe clinical symptoms and higher viral loads upon IAV infection. Mechanistically, BAG6 restricted IAV transcription and replication by inhibiting the activity of viral RNA-dependent RNA polymerase (RdRp). The co-immunoprecipitation assays showed BAG6 specifically interacted with the N-terminus of PB2 and competed with PB1 for RdRp complex assembly. The ubiquitination assay indicated that BAG6 promoted PB2 ubiquitination at K189 residue and targeted PB2 for K48-linked ubiquitination degradation. The antiviral effect of BAG6 necessitated its N-terminal region containing a ubiquitin-like (UBL) domain (17-92aa) and a PB2-binding domain (124-186aa), which are synergistically responsible for viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly. These findings unravel a novel antiviral mechanism via the interaction of viral PB2 and host protein BAG6 during avian or human influenza virus infection and highlight a potential application of BAG6 for antiviral drug development.

Navigating the Immune Response Landscape of Hepatitis B and Hepatitis C Virus Infections

Both the hepatitis B virus (HBV) and the hepatitis C virus (HCV) are viral pathogens that primarily target the liver and can lead to chronic liver disease. The nature of the immune response that the host mounts can vary depending on the structural differences between these two viruses. HBV is a partially double-stranded DNA virus enveloped in an outer envelope with a relatively stable genome and limited antigenic variation. It consists of an inner nucleocapsid core containing viral DNA, viral polymerase, and core antigen (HBcAg). The envelope comprises surface antigens (HBsAg) important for viral entry and immune recognition. HCV is a single-stranded RNA virus with an envelope. It has a high degree of genetic diversity due to its error-prone RNA polymerase, resulting in multiple HCV genotypes and subtypes due to its high mutation rate. Both viruses activate innate immune responses, producing type I interferons (IFNs) and pro-inflammatory cytokines. HBV can actively fight these antiviral responses in several ways, such as by making viral proteins that mess up the innate immune signalling pathways. At the same time, HCV developed strategies to evade and modulate the host's innate immune system, allowing it to establish persistent infections. The adaptive immune response against HBV and HCV involves both humoral and cellular components. Antibodies against HBsAg (anti-HBs) are critical for viral clearance and protection. CD8+ T cells are also very important for controlling HBV infection because they find and kill infected hepatocytes. During infection, HCV-specific antibodies and CD4+ and CD8+ T cells are made. However, HCV can evade immune responses by rapidly mutating its surface proteins (e.g., E2) and modulating T-cell responses. It is important to note that the immune response to HBV and HCV is a complex and dynamic process that involves various factors beyond the structural differences described above. Host factors, viral load, viral persistence, and the interplay between innate and adaptive immune responses also significantly influence the outcome of the infection

Chicken UFL1 Restricts Avian Influenza Virus Replication by Disrupting the Viral Polymerase Complex and Facilitating Type I IFN Production.

During avian influenza virus (AIV) infection, host defensive proteins promote antiviral innate immunity or antagonize viral components to limit viral replication. UFM1-specific ligase 1 (UFL1) is involved in regulating innate immunity and DNA virus replication in mammals, but the molecular mechanism by which chicken (ch)UFL1 regulates AIV replication is unclear. In this study, we first identified chUFL1 as a negative regulator of AIV replication by enhancing innate immunity and disrupting the assembly of the viral polymerase complex. Mechanistically, chUFL1 interacted with chicken stimulator of IFN genes (chSTING) and contributed to chSTING dimerization and the formation of the STING-TBK1-IRF7 complex. We further demonstrated that chUFL1 promoted K63-linked polyubiquitination of chSTING at K308 to facilitate chSTING-mediated type I IFN production independent of UFMylation. Additionally, chUFL1 expression was upregulated in response to AIV infection. Importantly, chUFL1 also interacted with the AIV PA protein to inhibit viral polymerase activity. Furthermore, chUFL1 impeded the nuclear import of the AIV PA protein and the assembly of the viral polymerase complex to suppress AIV replication. Collectively, these findings demonstrate that chUFL1 restricts AIV replication by disrupting the viral polymerase complex and facilitating type I IFN production, which provides new insights into the regulation of AIV replication in chickens.

Structural characterization of the oligomerization of full-length Hantaan virus polymerase into symmetric dimers and hexamers

Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. Here we describe the complete cryo-electron microscopy structure of Hantaan virus polymerase in several oligomeric forms. Apo polymerase protomers can adopt two drastically different conformations, which assemble into two distinct symmetric homodimers, that can themselves gather to form hexamers. Polymerase dimerization induces the stabilization of most polymerase domains, including the C-terminal domain that contributes the most to dimer’s interface, along with a lariat region that participates to the polymerase steadying. Binding to viral RNA induces significant conformational changes resulting in symmetric oligomer disruption and polymerase activation, suggesting the possible involvement of apo multimers as protecting systems that would stabilize the otherwise flexible C-terminal domains. Overall, these results provide insights into the multimerization capability of Hantavirus polymerase and may help to define antiviral compounds to counteract these life-threatening viruses.

Structural Impact of the Interaction of the Influenza A Virus Nucleoprotein with Genomic RNA Segments.

Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions.

Utilising organs from hepatitis C virus PCR-positive donors in Western Australia.

We transplanted six solid organs from three hepatitis C virus (HCV) polymerase chain reaction (PCR)-positive donors during 2018-2023. Recipients were treated with glecaprevir/pibrentasvir or sofosbuvir/velpatasvir for 4-12 weeks, with all six achieving sustained virological response without significant adverse events. As occurs in other jurisdictions, solid organ transplants from HCR PCR-positive donors can be safely utilised in Australia.

BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir.

Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128's primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently-but complementary-with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect-a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

The conserved RNP motif of the herpes simplex virus 1 family B DNA polymerase is crucial for viral DNA synthesis but not polymerase activity

The herpes simplex virus 1 DNA polymerase contains a highly conserved structural motif found in most family B polymerases and certain RNA-binding proteins. To investigate its importance within cells, we constructed a mutant virus with substitutions in two residues of the motif and a rescued derivative. The substitutions resulted in severe impairment of plaque formation, yields of infectious virus, and viral DNA synthesis while not meaningfully affecting expression of the mutant enzyme, its co-localization with the viral single-stranded DNA binding protein at intranuclear punctate sites in non-complementing cells or in replication compartments in complementing cells, or viral DNA polymerase activity. Taken together, our results indicate that the RNA binding motif plays a crucial role in herpes simplex virus 1 DNA synthesis through a mechanism separate from effects on polymerase activity, thus identifying a distinct essential function of this motif with implications for hypotheses regarding its biochemical functions.

PB2 residue 473 contributes to the mammalian virulence of H7N9 avian influenza virus by modulating viral polymerase activity via ANP32A.

Since the first human infection reported in 2013, H7N9 avian influenza virus (AIV) has been regarded as a serious threat to human health. In this study, we sought to identify the virulence determinant of the H7N9 virus in mammalian hosts. By comparing the virulence of the SH/4664 H7N9 virus, a non-virulent H9N2 virus, and various H7N9-H9N2 hybrid viruses in infected mice, we first pinpointed PB2 as the primary viral factor accounting for the difference between H7N9 and H9N2 in mammalian virulence. We further analyzed the in vivo effects of individually mutating H7N9 PB2 residues different from the closely related H9N2 virus and consequently found residue 473, alongside the well-known residue 627, to be critical for the virulence of the H7N9 virus in mice and the activity of its reconstituted viral polymerase in mammalian cells. The importance of PB2-473 was further strengthened by studying reverse H7N9 substitutions in the H9N2 background. Finally, we surprisingly found that species-specific usage of ANP32A, a family member of host factors connecting with the PB2-627 polymorphism, mediates the contribution of PB2 473 residue to the mammalian adaption of AIV polymerase, as the attenuating effect of PB2 M473T on the viral polymerase activity and viral growth of the H7N9 virus could be efficiently complemented by co-expression of chicken ANP32A but not mouse ANP32A and ANP32B. Together, our studies uncovered the PB2 473 residue as a novel viral host range determinant of AIVs via species-specific co-opting of the ANP32 host factor to support viral polymerase activity.IMPORTANCEThe H7N9 avian influenza virus has been considered to have the potential to cause the next pandemic since the first case of human infection reported in 2013. In this study, we identified PB2 residue 473 as a new determinant of mouse virulence and mammalian adaptation of the viral polymerase of the H7N9 virus and its non-pathogenic H9N2 counterparts. We further demonstrated that the variation in PB2-473 is functionally linked to differential co-opting of the host ANP32A protein in supporting viral polymerase activity, which is analogous to the well-known PB2-627 polymorphism, albeit the two PB2 positions are spatially distant. By providing new mechanistic insight into the PB2-mediated host range determination of influenza A viruses, our study implicated the potential existence of multiple PB2-ANP32 interfaces that could be targets for developing new antivirals against the H7N9 virus as well as other mammalian-adapted influenza viruses.

Ebola virus VP35 interacts non-covalently with ubiquitin chains to promote viral replication.

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome.

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

Characterization of SLA RNA promoter from dengue virus and its interaction with the viral non-structural NS5 protein

The Dengue virus (DENV) is the most significant arthropod-borne viral pathogen in humans with 400 million infections annually. DENV comprises four distinct serotypes (DENV-1 to -4) which complicates vaccine development. Any of the four serotypes can cause clinical illness but with distinctive infection dynamics. Variations in sequences identified within the four genomes induce structural differences in crucial RNA motifs that were suggested to be correlated to the degree of pathogenicity among DENV-1 to -4. In particular, the RNA Stem-loop A (SLA) at the 5′-end of the genome, acts as a key regulator of the viral replication cycle by interacting with the viral NS5 polymerase to initiate the minus-strand viral RNA synthesis and later to methylate and cap the synthesized RNA. The molecular details of this interaction remain not fully described. Here, we report the solution secondary structures of SLA from DENV-1 to -4. Our results highlight that the four SLA exhibit structural and dynamic differences. Secondly, to determine whether SLA RNA contains serotype-specific determinants for the recognition by the viral NS5 protein, we investigated interactions between SLA from DENV -1 to -4 and DENV2 NS5 using combined biophysical approaches. Our results show that NS5 from DENV2 is able to bind SLA from other serotypes, but that other viral or host factors may be necessary to stabilize the complex and promote the catalytically active state of the NS5. By contrast, we show that a serotype-specific binding is driven by specific interactions involving conformational changes within the SLA RNA.

Unravelling the genomic origins of lumpy skin disease virus in recent outbreaks.

Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus and family Poxviridae. LSDV was endemic in most of Africa, the Middle East and Turkey, but since 2015, several outbreaks have been reported in other countries. In this study, we used whole genome sequencing approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations compared to the Reference Neethling strain sequence and the previous field strains. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. Thirty-eight genes have more than 2 variations per gene, and these genes belong to viral-core proteins, viral binding proteins, replication, and RNA polymerase proteins. We highlight the importance of several SNPs in various genes, which may play an essential role in the pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues.

Pulmonary toxicity associated with high-dose favipiravir and treatment options: biochemical and histopathological evaluation.

Favipiravir is a broad-spectrum antiviral drug that is a viral RNA-dependent RNA polymerase inhibitor. Favipiravir is used in high doses to treat COVID-19 but has a side effect on humans at high doses. The side effects of favipiravir have been associated with oxidative stress in the literature. In this trial, we investigated the biochemical and histopathological effects of lacidip-ine, thiamine pyrophosphate (TTP), and adenosine triphosphate (ATP), drugs with antioxidant properties, on the lung toxicity caused by high-dose favipiravir in rats. The rats were classified into five groups: healthy (HG), favipiravir alone (Fav), lacidipine+favipiravir (LFav), TPP+favipiravir (TFav), and ATP+favipiravir (AFav). Favipiravir (800 mg/kg) was administered twice daily for seven days. Laci-dipine (4 mg/kg), TPP (20 mg/kg), and ATP (25 mg/kg) were administered once daily for seven days. Oxidant (malondialdehyde), non-enzymatic (total glutathi-one), and enzymatic (superoxide dismutase and catalase) antioxidant levels were measured in the excised lung tissues. Furthermore, the tissues were histopatho-logically examined. The systemic administration of high doses of favipiravir in-creased oxidant levels and decreased antioxidant levels in the lung tissue of rats. In parallel, the histopathological examination of the lung tissue revealed the presence of severe mononuclear cell infiltrations in interstitial areas and pronounced lymphoid hyperplasia. Lacidipine exhibited superior efficacy in mit-igating oxidative stress and preventing the decline of antioxidants induced by favipiravir compared with TPP and ATP. Histopathologically, the lacidipine admin-istration significantly reduced lung oxidative damage. TTP moderately reduced severe favipiravir-associated lung injury. However, ATP was ineffective against fa-vipiravir-associated lung injury. Lacidipine offers more therapeutic benefits than TPP in treating oxidative lung injury caused by high doses of favipiravir.

Nairovirus polymerase mutations associated with the establishment of persistent infection in human cells.

We investigated whether and how nairoviruses persistently infect human cells, using Hazara orthonairovirus (HAZV), a surrogate model for Crimean-Congo hemorrhagic fever virus. We established a human cell line that was persistently infected with HAZV. Five mutations were found in L protein of persistently infected HAZV (HAZVpi): mut1, mut2, mut3, mut4, and mut5. Among them, Lmut1 and Lmut4 restricted viral growth by low polymerase activity and low growth rate, respectively, leading to inhibition of viral overgrowth. The restriction of viral growth caused by Lmut1 and Lmut4 was compensated by other mutations, including Lmut2, Lmut3, and Lmut5. Each of the mutations found in L protein of HAZVpi was concluded to cooperatively modulate viral growth, which facilitates the establishment of persistent infection. Suppression of innate immunity also potentially contributes to virus persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells.