A novel ormycovirus isolated from the plant-pathogenic fungus Fusarium graminearum.

In this study, we identified a novel mycovirus, Fusarium graminearum ormycovirus 1 (FgOV1), from the pathogenic fungus Fusarium graminearum. The virus has two RNA segments, RNA1 and RNA2, with lengths of 2,591 and 1,801 nucleotides, respectively, excluding the polyA tail. Each segment contains a single open reading frame (ORF). The ORF in RNA1 encodes an RNA-dependent RNA polymerase, while the ORF in RNA2 encodes a hypothetical protein. Phylogenetic analysis showed that FgOV1 belongs to the gammaormycovirus clade, whose members are related to betaormycoviruses. To our knowledge, this is the first report of an ormycovirus in Fusarium graminearum.

Silencing of RDR1 and RDR6 genes by a single RNAi enhances lettuce's capacity to express recombinant proteins in transient assays.

KEY MESSAGE: Enhanced recombinant protein expression was achieved in Salinas lettuce and commercial lettuce by designing a unique RNAi that knockdown the gene-silencing mechanism in transient assays. Improved yields of recombinant proteins (RP) are necessary for protein-production efficiency and ease of purification. Achieving high yield in non-tobacco plants will enable diverse plants to be used as hosts in transient protein-expression systems. With improved protein yield, lettuce (Lactuca sativa) could take the lead as a plant host for RP production. Therefore, this study aimed to improve RP production in lettuce var. Salinas by designing a single RNA interference (RNAi) construct targeting LsRDR1 and LsRDR6 using the Tsukuba system vector. Two RNAi constructs, RNAi-1 and RNAi-2, targeting common regions of LsRDR1 and LsRDR6 with 75% and 76% similarity, respectively, were employed to evaluate simultaneous gene silencing. Quantitative transcription analysis demonstrated that both RNAi constructs effectively knocked down LsRDR6 and LsRDR1, but not LsRDR2, at both 3 and 5 days post-infiltration (dpi), with RNAi-1 exhibited slightly higher efficiency. Based on the protein yield, co-expression of RNAi-1 with enhanced green fluorescent protein (EGFP) increased EGFP expression by approximately 4.9-fold and 3.7-fold at 3 dpi and 5 dpi, respectively, compared to control. A similar but slightly lower increase (2.4-fold and 2.33-fold) was observed in commercial lettuce at 3 and 5 dpi, respectively. To confirm these results, co-infiltration with Bet v 1, a major allergen from birch pollen, resulted in a 2.5-fold increase in expression in Salinas lettuce at 5 dpi. This study marks a significant advancement in enhancing transient protein production in lettuce, elevating its potential as a host for recombinant protein production.

Discovery and genomic characterization of three double-stranded RNA viruses coinfecting Conidiobolus taihushanensis.

Conidiobolus sensu lato, a genus within the family Ancylistaceae, encompasses a diverse range of fungal species that are widely distributed in plant debris and soil. In this study, we identified three double-stranded RNA (dsRNA) viruses coinfecting a strain of Conidiobolus taihushanensis. These viruses were identified as Conidiobolus taihushanensis totivirus 1 (CtTV1), Conidiobolus nonsegmented RNA virus 1-2 (CNRV1-2), and Conidiobolus taihushanensis virus 1 (CtV1). Through high-throughput sequencing and RNA-ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we determined their complete genome sequences. The genome of CtTV1 is 6,921 nucleotides in length, containing two open reading frames (ORFs). ORF1 encodes a 1,124-amino-acid capsid protein (CP) with a molecular weight of 125.07 kDa, and ORF2 encodes a 780-amino-acid RNA-dependent RNA polymerase (RdRp) with a molecular weight of 88.05 kDa. CNRV1-2, approximately 3.0 kb in length, also contains two ORFs, which are predicted to encode a 186-amino-acid hypothetical protein (HP) and a 758-amino-acid RdRp. CtV1 has a smaller genome consisting of 3,081 base pairs (bp) with two ORFs: one encoding a 244-amino-acid HP (26.85 kDa) and the other encoding a 707-amino-acid RdRp (80.64 kDa). Phylogenetic analysis based on RdRp sequences revealed that CtTV1 shows the highest similarity to Phytophthora pluvialis RNA virus 1, with 38.79% sequence identity, and clusters with members of the family Orthototiviridae, and it is most closely related to Utsjoki toti-like virus. In contrast, CtV1 formed a unique branch and might represent a new genus. The genome sequence of CNRV1-2 is 99.74% identical to that of the previously described Conidiobolus non-segmented RNA virus 1 (CNRV1). Our findings indicate that CtTV1 and CtV1 are distinct novel viruses, while CNRV1-2 appears to be a variant of CNRV1. This study enhances our understanding of the genetic diversity and evolutionary relationships among mycoviruses associated with C. taihushanensis.

Towards the Development of a Minigenome Assay for Species A Rotaviruses.

RNA virus polymerases carry out multiple functions necessary for successful genome replication and transcription. A key tool for molecular studies of viral RNA-dependent RNA polymerases (RdRps) is a 'minigenome' or 'minireplicon' assay, in which viral RdRps are reconstituted in cells in the absence of full virus infection. Typically, plasmids expressing the viral polymerase protein(s) and other co-factors are co-transfected, along with a plasmid expressing an RNA encoding a fluorescent or luminescent reporter gene flanked by viral untranslated regions containing cis-acting elements required for viral RdRp recognition. This reconstitutes the viral transcription/replication machinery and allows the viral RdRp activity to be measured as a correlate of the reporter protein signal. Here, we report on the development of a 'first-generation' plasmid-based minigenome assay for species A rotavirus using a firefly luciferase reporter gene.

Prediction of promiscuous multiepitope-based peptide vaccine against RdRp of rotavirus using immunoinformatics studies.

Rotavirus, a dsRNA virus in the Reoviridae family, shows a segmented genome. The VP1 gene encodes the RNA-dependent RNA polymerase (RdRp). This study aims to develop a multiepitope-based vaccine targeting RdRp using immunoinformatic approaches. In this study, 100 available nucleotide sequences of VP1-Rotavirus belonging to different strains across the world were retrieved from NCBI database. The selected sequences were aligned, and a global consensus sequence was developed by using CLC work bench. The study involved immunoinformatic approaches and molecular docking studies to reveal the promiscuous epitopes that can be eventually used as active vaccine candidates for Rotavirus. In total, 27 highly immunogenic, antigenic, and non-allergenic T-cell and B-cell epitopes were predicted for the Multiepitope vaccine (MEV) against rotavirus. It was also observed that MEV can prove to be effective worldwide due to its high population coverage, demonstrating the consistency of this vaccine. Moreover, there is a high docking interaction and immunological response with a binding score of -50.2 kcal/mol, suggesting the vaccine's efficacy. Toll-like receptors (TLRs) also suggest that the vaccine is physiologically and immunologically effective. Collectively, our data point to an effective MEV against rotavirus that can effectively reduce viral infections and improve the health status worldwide.

Two +ssRNA mycoviruses cohabiting the fungal cultivar of leafcutter ants.

Leafcutter ants are dominant herbivores in the Neotropics and rely on a fungus (Leucoagaricus gongylophorus) to transform freshly gathered leaves into a source of nourishment rather than consuming the vegetation directly. Here we report two virus-like particles that were isolated from L. gongylophorus and observed using transmission electron microscopy. RNA sequencing identified two +ssRNA mycovirus strains, Leucoagaricus gongylophorus tymo-like virus 1 (LgTlV1) and Leucoagaricus gongylophorus magoulivirus 1 (LgMV1). Genome annotation of LgTlV1 (7401 nt) showed conserved domains for methyltransferase, endopeptidase, viral RNA helicase, and RNA-dependent RNA polymerase (RdRp). The smaller genome of LgMV1 (2636 nt) contains one open reading frame encoding an RdRp. While we hypothesize these mycoviruses function as symbionts in leafcutter farming systems, further study will be needed to test whether they are mutualists, commensals, or parasites.

Detection and molecular characterization of a novel mitovirus associated with Passiflora edulis Sims.

Mitoviruses are cryptic capsidless viruses belonging to the family Mitoviridae that replicate and are maintained in the mitochondria of fungi. Complete mitovirus-like sequences were recently assembled from plant transcriptome data and plant leaf tissue samples. Passion fruit (Passiflora spp.) is an economically important crop for numerous tropical and subtropical countries worldwide, and many virus-induced diseases impact its production. From a large-scale genomic study targeting viruses infecting Passiflora spp. in Brazil, we detected a de novo-assembled contig with similarity to other plant-associated mitoviruses. The contig is ∼2.6 kb long, with a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRP). This contig has been named "passion fruit mitovirus-like 1" (PfMv1). An alignment of the predicted amino acid sequence of the RdRP of PfMv1 and those of other plant-associated mitoviruses revealed the presence of the six conserved motifs of mitovirus RdRPs. PfMv1 has 79% coverage and 50.14% identity to Humulus lupulus mitovirus 1. Phylogenetic analysis showed that PfMV1 clustered with other plant-associated mitoviruses in the genus Duamitovirus. Using RT-PCR, we detected a PfMv1-derived fragment, but no corresponding DNA was identified, thus excluding the possibility that this is an endogenized viral-like sequence. This is the first evidence of a replicating mitovirus associated with Passiflora edulis, and it should be classified as a member of a new species, for which we propose the name "Duamitovirus passiflorae".

Complete genome sequence of lima bean endornavirus 1: a putative new member of the genus Alphaendornavirus (family Endornaviridae).

In this study, we completely sequenced the genome of a new member of the genus Alphaendornavirus, family Endornaviridae, from lima bean (Phaseolus lunatus), for which we propose the name "lima bean endornavirus 1" (LbEV1). The complete genome of LbEV1 consists of 15,265 nucleotides, including a stretch of 12 cytosine residues at its 3' end, and contains a long single open reading frame (ORF) coding for a 4980-aa-long polyprotein. Analysis of the polyprotein sequence revealed the presence of four conserved functional domains (in order from the N- to C-terminus): viral helicase 1, peptidase _C97, glycosyltransferase_GTB-type, and viral RNA-dependent RNA polymerase (RdRP). The LbEV1 polyprotein showed the highest amino acid sequence similarity (63% identity and 98% coverage) to Phaseolus vulgaris endornavirus 3 (PvEV3) and also showed 42% identity (95% coverage) to Geranium carolinianum endornavirus. Phylogenetic analysis based on the viral RdRp domain showed that LbEV1 belongs to a subclade within the genus Alphaendornavirus that includes three other viruses infecting plants of the genus Phaseolus.

Structures of influenza A and B replication complexes give insight into avian to human host adaptation and reveal a role of ANP32 as an electrostatic chaperone for the apo-polymerase.

Replication of influenza viral RNA depends on at least two viral polymerases, a parental replicase and an encapsidase, and cellular factor ANP32. ANP32 comprises an LRR domain and a long C-terminal low complexity acidic region (LCAR). Here we present evidence suggesting that ANP32 is recruited to the replication complex as an electrostatic chaperone that stabilises the encapsidase moiety within apo-polymerase symmetric dimers that are distinct for influenza A and B polymerases. The ANP32 bound encapsidase, then forms the asymmetric replication complex with the replicase, which is embedded in a parental ribonucleoprotein particle (RNP). Cryo-EM structures reveal the architecture of the influenza A and B replication complexes and the likely trajectory of the nascent RNA product into the encapsidase. The cryo-EM map of the FluB replication complex shows extra density attributable to the ANP32 LCAR wrapping around and stabilising the apo-encapsidase conformation. These structures give new insight into the various mutations that adapt avian strain polymerases to use the distinct ANP32 in mammalian cells.

Characterization of viroplasm-like structures by co-expression of NSP5 and NSP2 across rotavirus species A to J.

Rotaviruses (RVs) are classified into nine species, A-D and F-J, with species A being the most studied. In rotavirus of species A (RVA), replication occurs in viroplasms, which are cytosolic globular inclusions composed of main building block proteins NSP5, NSP2, and VP2. The co-expression of NSP5 with either NSP2 or VP2 in uninfected cells leads to the formation of viroplasm-like structures (VLSs). Although morphologically identical to viroplasms, VLSs do not produce viral progeny but serve as excellent tools for studying complex viroplasms. A knowledge gap exists regarding non-RVA viroplasms due to the lack of specific antibodies and suitable cell culture systems. In this study, we explored the ability of NSP5 and NSP2 from non-RVA species to form VLSs. The co-expression of these two proteins led to globular VLSs in RV species A, B, D, F, G, and I, while RVC formed filamentous VLSs. The co-expression of NSP5 and NSP2 of RV species H and J did not result in VLS formation. Interestingly, NSP5 of all RV species self-oligomerizes, with the ordered C-terminal region, termed the tail, being necessary for self-oligomerization of RV species A-C and G-J. Except for NSP5 from RVJ, all NSP5 interacted with their cognate NSP2. We also found that interspecies VLS are formed between closely related RV species B with G and D with F. Additionally, VLS from RVH and RVJ formed when the tail of NSP5 RVH and RVJ was replaced by the tail of NSP5 from RVA and co-expressed with their respective NSP2. IMPORTANCE: Rotaviruses (RVs) are classified into nine species, A-D and F-J, infecting mammals and birds. Due to the lack of research tools, all cumulative knowledge on RV replication is based on RV species A (RVA). The RV replication compartments are globular cytosolic structures named viroplasms, which have only been identified in RV species A. In this study, we examined the formation of viroplasm-like structures (VLSs) by the co-expression of NSP5 with NSP2 across RV species A to J. Globular VLSs formed for RV species A, B, D, F, G, and I, while RV species C formed filamentous structures. The RV species H and J did not form VLS with their cognates NSP5 and NSP2. Similar to RVA, NSP5 self-oligomerizes in all RV species, which is required for VLS formation. This study provides basic knowledge of the non-RVA replication mechanisms, which could help develop strategies to halt virus infection across RV species.

Transcription elongation of the plant RNA polymerase IV is prone to backtracking.

RNA polymerase IV (Pol IV) forms a complex with RNA-directed RNA polymerase 2 (RDR2) to produce double-stranded RNA (dsRNA) precursors essential for plant gene silencing. In the "backtracking-triggered RNA channeling" model, Pol IV backtracks and delivers its transcript's 3' terminus to RDR2, which synthesizes dsRNA. However, the mechanisms underlying Pol IV backtracking and RNA protection from cleavage are unclear. Here, we determined cryo-electron microscopy structures of Pol IV elongation complexes at four states of its nucleotide addition cycle (NAC): posttranslocation, guanosine triphosphate-bound, pretranslocation, and backtracked states. The structures reveal that Pol IV maintains an open DNA cleft and kinked bridge helix in all NAC states, loosely interacts with the nucleoside triphosphate substrate, and barely contacts proximal backtracked nucleotides. Biochemical data indicate that Pol IV is inefficient in forward translocation and RNA cleavage. These findings suggest that Pol IV transcription elongation is prone to backtracking and incapable of RNA hydrolysis, ensuring efficient dsRNA production by Pol IV-RDR2.

Molecular characterization of a novel gammapartitivirus infecting the fungus Nigrospora oryzae.

Here, we identified a new mycovirus infecting the phytopathogenic fungus Nigrospora oryzae, which we have designated "Nigrospora oryzae partitivirus 2" (NoPV2). The genome of NoPV2 consists of two dsRNA segments (dsRNA 1 and dsRNA 2), measuring 1771 and 1440 bp in length, respectively. dsRNA 1 and dsRNA 2 each contain a single open reading frame (ORF) that encodes the RNA-dependent RNA polymerase (RdRp) and capsid protein (CP), respectively. A BLASTp search showed that the RdRp of NoPV2 had significant sequence similarity to the RdRps of other partitiviruses, including Nigrospora sphaerica partitivirus 1 (75.61% identity) and Magnaporthe oryzae partitivirus 1 (67.53% identity). Phylogenetic analysis revealed that NoPV2 is a new member of the genus Gammapartitivirus in the family Partitiviridae. This study provides important information for understanding the diversity of mycoviruses in N. oryzae.

Complete genome sequence of a novel mitovirus isolated from the phytopathogenic fungus Alternaria alternata causing apple leaf blotch.

In this study, a novel mitovirus, tentatively designated as "Alternaria alternata mitovirus 2" (AaMV2), was isolated from the fungus Alternaria alternata f. sp. mali causing apple leaf blotch disease. The complete genome of AaMV2 is 3,157 nucleotides in length, with an A+U content of 68.10%. The genome has a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) protein with a molecular mass of 98.10 kDa. BLAST analysis revealed that AaMV2 has the highest sequence identity to Leptosphaeria biglobosa mitovirus 6, with 79.76% and 82.86% identity at the amino acid and nucleotide level, respectively. Phylogenetic analysis suggested that AaMV2 is a new member of the genus Duamitovirus within the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus in A. alternata.

Molecular characterization of a novel mitovirus from the plant-pathogenic fungus Nigrospora oryzae.

Here, we characterized a novel mitovirus from the fungus Nigrospora oryzae, which was named "Nigrospora oryzae mitovirus 3" (NoMV3). The NoMV3 genome is 2,492 nt in length with a G + C content of 33%, containing a single large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes an RNA-dependent RNA polymerase (RdRp) of 775 amino acids with a molecular mass of 88.75 kDa. BLASTp analysis revealed that the RdRp of NoMV3 had 68.6%, 50.6%, and 48.6% sequence identity to those of Nigrospora oryzae mitovirus 2, Suillus luteus mitovirus 6, and Fusarium proliferatum mitovirus 3, respectively, which belong to the genus Unuamitovirus within the family Mitoviridae. Phylogenetic analysis based on amino acid sequences supported the classification of NoMV3 as a member of a new species in the genus Unuamitovirus within the family Mitoviridae.

First Report on Detection and Molecular Characterization of Astroviruses in Mongooses.

Applying a pan-astrovirus (AstV) RT-hemi-nested PCR assay, we report here high detection rates (28.3%, 15/53) of AstVs in the small Indian mongoose (Urva auropunctata) on the Caribbean Island of St. Kitts. Based on deduced amino acid (aa) identities and phylogenetic analysis of long RNA-dependent RNA polymerase (RdRp) sequences (~315 aa, partial RdRp), the AstVs detected in the mongooses (designated as Mon-AstVs) were classified into two distinct groups (deduced aa identities of 66.45-67.30% between the groups). The putative RdRps of the Mon-AstVs shared low deduced aa identities with those of AstVs from other host species (<69%, <54%, and <50% identities with reptilian/amphibian AstVs, avastroviruses, and mamastroviruses, respectively). Phylogenetically, the group-I and group-II Mon-AstVs formed two distinct clusters, near the cluster of reptilian/amphibian AstVs, and were distantly related to avastroviruses and mamastroviruses. Since the mongooses were apparently healthy during sampling, we could not establish if the Mon-AstVs infected the animal or were of dietary origin. Although we could not ascertain the true host of the Mon-AstVs, phylogenetic analysis indicated that these viruses might have originated from lower vertebrates. To our knowledge, this is the first report on the detection and molecular characterization of AstVs in mongooses, highlighting the wide host range and significant genetic diversity within the family Astroviridae.

Two thiosemicarbazones derived from 1-indanone as potent non-nucleoside inhibitors of bovine viral diarrhea virus of different genotypes and biotypes.

Bovine viral diarrhea virus (BVDV) is a widespread pathogen of cattle and other mammals that causes major economic losses in the livestock industry. N4-TSC and 6NO2-TSC are two thiosemicarbazones derived from 1-indanone that exhibit anti-BVDV activity in vitro. These compounds selectively inhibit BVDV and are effective against both cytopathic and non-cytopathic BVDV-1 and BVDV-2 strains. We confirmed that N4-TSC acts at the onset of viral RNA synthesis, as previously reported for 6NO2-TSC. Moreover, resistance selection and characterization showed that N4-TSCR mutants were highly resistant to N4-TSC but remained susceptible to 6NO2-TSC. In contrast, 6NO2-TSCR mutants were resistant to both compounds. Additionally, mutations N264D and A392E were found in the viral RNA-dependent RNA polymerase (RdRp) of N4-TSCR mutants, whereas I261 M was found in 6NO2-TSCR mutants. These mutations lay in a hydrophobic pocket within the fingertips region of BVDV RdRp that has been described as a "hot spot" for BVDV non-nucleoside inhibitors.

The PB2 I714S mutation influenced mammalian adaptation of the H3N2 canine influenza virus by interfering with nuclear import efficiency and RNP complex assembly.

Avian influenza viruses (AIVs) are the origin of multiple mammal influenza viruses. The genetic determinants of AIVs adapted to humans have been widely elucidated, however, the molecular mechanism of cross-species transmission and adaptation of AIVs to canines are still poorly understood. In this study, two H3N2 influenza viruses isolated from a live poultry market (A/environment/Guangxi/13431/2018, GX13431) and a swab sample from a canine (A/canine/Guangdong/0601/2019, GD0601) were used to investigate the possible molecular basis that determined H3N2 AIV adapting to canine. We found that GD0601 exhibited more robust polymerase activity in cells and higher pathogenicity in mice compared with its evolution ancestor H3N2 AIV GX13431. A series of reassortments of the ribonucleoprotein (RNP) complex showed that the PB2 subunit was the crucial factor that conferred high polymerase activity of GD0601, and the substitution of I714S in the PB2 subunit of GD0601 attenuated the replication and pathogenicity in mammal cells and the mouse model. Mechanistically, the reverse mutation of I714S in the PB2 polymerase subunit which was identified in AIV GX13431 reduced the nuclear import efficiency of PB2 protein and interfered with the interactions of PB2-PA/NP that affected the assembly of the viral RNP complex. Our study reveals amino acid mutation at the position of 714 in the nuclear localization signal (NLS) area in PB2 plays an important role in overcoming the barrier from poultry to mammals of the H3N2 canine influenza virus and provides clues for further study of mammalian adaptation mechanism of AIVs.

Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase.

Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

Detection of influenza A(H3N2) viruses with polymerase acidic subunit substitutions after and prior to baloxavir marboxil treatment during the 2022-2023 influenza season in Japan.

Baloxavir marboxil (baloxavir), approved as an anti-influenza drug in Japan in March 2018, can induce reduced therapeutic effectiveness due to PA protein substitutions. We assessed PA substitutions in clinical samples from influenza-infected children and adults pre- and post-baloxavir treatment, examining their impact on fever and symptom duration. During the 2022-2023 influenza season, the predominant circulating influenza subtype detected by cycling-probe RT-PCR was A(H3N2) (n = 234), with a minor circulation of A(H1N1)pdm09 (n = 10). Of the 234 influenza A(H3N2) viruses collected prior to baloxavir treatment, 2 (0.8%) viruses carry PA/I38T substitution. One virus was collected from a toddler and one from an adult, indicating the presence of viruses with reduced susceptibility to baloxavir, without prior exposure to the drug. Of the 54 paired influenza A(H3N2) viruses collected following baloxavir treatment, 8 (14.8%) viruses carried E23 K/G, or I38 M/T substitutions in PA. Variant calling through next-generation sequencing (NGS) showed varying proportions (6-100 %), a polymorphism and a mixture of PA/E23 K/G, and I38 M/T substitutions in the clinical samples. These eight viruses were obtained from children aged 7-14 years, with a median fever duration of 16.7 h and a median symptom duration of 93.7 h, which were similar to those of the wild type. However, the delayed viral clearance associated with the emergence of PA substitutions was observed. No substitutions conferring resistance to neuraminidase inhibitors were detected in 37 paired samples collected before and following oseltamivir treatment. These findings underscore the need for ongoing antiviral surveillance, informing public health strategies and clinical antiviral recommendations for seasonal influenza.

High throughput profiling identified PA-L106R amino acid substitution in A(H1N1)pdm09 influenza virus that confers reduced susceptibility to baloxavir in vitro.

Baloxavir acid (BXA) is a pan-influenza antiviral that targets the cap-dependent endonuclease of the polymerase acidic (PA) protein required for viral mRNA synthesis. To gain a comprehensive understanding on the molecular changes associated with reduced susceptibility to BXA and their fitness profile, we performed a deep mutational scanning at the PA endonuclease domain of an A (H1N1)pdm09 virus. The recombinant virus libraries were serially passaged in vitro under increasing concentrations of BXA followed by next-generation sequencing to monitor PA amino acid substitutions with increased detection frequencies. Enriched PA amino acid changes were each introduced into a recombinant A (H1N1)pdm09 virus to validate their effect on BXA susceptibility and viral replication fitness in vitro. The I38 T/M substitutions known to confer reduced susceptibility to BXA were invariably detected from recombinant virus libraries within 5 serial passages. In addition, we identified a novel L106R substitution that emerged in the third passage and conferred greater than 10-fold reduced susceptibility to BXA. PA-L106 is highly conserved among seasonal influenza A and B viruses. Compared to the wild-type virus, the L106R substitution resulted in reduced polymerase activity and a minor reduction of the peak viral load, suggesting the amino acid change may result in moderate fitness loss. Our results support the use of deep mutational scanning as a practical tool to elucidate genotype-phenotype relationships, including mapping amino acid substitutions with reduced susceptibility to antivirals.