Journal of General Virology

Since 2020, the United Kingdom (UK) has suffered repeated epizootics of clade 2.3.4.4b H5 high pathogenicity avian influenza viruses (HPAIVs) in wild birds and poultry, resulting in substantial economic losses due to enforced statutory control. The rapid evolution of H5 HPAIVs continues to raise concern with heightened zoonotic and pandemic risks. The immunodominant haemagglutinin glycoprotein (HA) is crucial for influenza virus receptor binding and pH-induced fusion of viral and cellular membranes. Mutations in HA are frequent due to polymerase error, immune pressure and host adaptation, resulting in antigenic modulation and/or an expansion of host tropism, respectively, ultimately hindering control strategies. We evaluated a comprehensive panel of H5 viruses representing prevalent genotypes from UK outbreaks spanning 2020 to 2022 for HA functionality. HA genes from each genotype were assessed through receptor binding, pH of fusion, thermostability and HA inhibition assays to evaluate factors contributing to zoonotic potential, stability, and antigenicity. The viruses only bound to avian receptors and exhibited fusion at a pH of 5.8, above the pH range (pH 5.0 to 5.5) associated with efficient human-to-human transmission. Therefore, these H5 viruses have low immediate zoonotic threat. Contemporary H5 viruses were more thermostable and showed antigenic drift compared to the earlier 2017-2018 clade 2.3.4.4b H5N8 viruses, and N236D in HA was identified as a significant antigenic epitope. The findings of this study underscore the evolving nature of the HA of these viruses and highlight the importance of ongoing surveillance and characterisation efforts to identify factors that might contribute to zoonotic risk.

The accumulation of subgenomic flavivirus RNAs (sfRNAs) modulates viral fitness and pathogenicity in culture and in vivo. These noncoding RNAs are produced by incomplete digestion of the flavivirus genome by the cellular 5-3 exoribonuclease (XRN1). Diverse flaviviruses have conserved RNA structural elements (RSEs) that map to their 3-untranslated region (3-UTR): Xrn-resistant RNA structures, dumbbell structures, and a 3-stem loop (3SL). Despite the importance of the 3-UTR RSEs for flavivirus replication, the structural dynamics of sfRNA during flavivirus infection are understudied. Here, we use digital droplet PCR to quantify sfRNA levels during infection for a panel of mosquito-borne flaviviruses (MbFV) including dengue virus serotypes 1 (DENV1), 2 (DENV2), and 4 (DENV4), and Zika virus (ZIKV). We then used SHAPE-MaP on XRN1-digested, in vitro-transcribed sfRNAs from each virus to determine their secondary structures compared to the corresponding sfRNAs obtained from flavivirus-infected A549 cells. Results seen in-cell and in vitro were largely similar; however, motifs within the dumbbell, the small hairpin (sHP) directly upstream of the 3-SL, and 3-SL regions showed significant differences in the extent of nucleotide reactivity. These differences were consistent among the four flaviviruses examined and may indicate regions of sfRNA that are shielded by interaction with proteins or other nucleic acids during infection. However, strong protection indicative of sustained interaction was not apparent. Our findings suggest that sfRNA interactions with viral and host factors within the cell are few, occur via base-paired regions, or are highly transient. ImportanceFlaviviruses are highly prevalent human pathogens. The flavivirus genome contains RNA structural elements (RSEs), including those encoded in the 3-UTR, that are necessary for viral replication. Subgenomic flavivirus RNAs (sfRNAs) are produced by incomplete digestion of flavivirus genomic RNA due to the cellular exoribonuclease XRN1 encountering 3-UTR RSEs that promote its stalling and disassociation. Viruses unable to produce sfRNAs are highly attenuated, underlining their biological importance. sfRNA secondary structure has been investigated previously but little information is available on sfRNA secondary structure dynamics in infected cells. By comparing SHAPE-MaP reactivities in vitro and in cells, we determined that previously inferred structures are likely maintained within infected cells. We also identified differences in the extent of SHAPE reactivity between in vitro and in-cell environments that were common to multiple mosquito-borne flaviviruses. These differences suggest that sfRNAs may engage in transient interactions within the cell that may be important for their function.

Dengue virus serotype 2 (DENV-2) is a mosquito-borne disease in the family Flaviviridae. It has been previously shown that DENV-2 can infect C6/36 mosquito cells and cause initial cytopathic effects that dissipate upon serial split-passage to yield persistently infected cultures with normal growth and morphology. In other words, the cell line accommodated persistent DENV-2 infections. It has recently been found that insect viral infections induce the production of viral copy DNA (vcDNA) fragments via host reverse transcriptase (RT). The vcDNA occurs in both linear (lvcDNA) and circular (cvcDNA) forms and produces small interfering RNA (siRNA) transcripts that can result in an immediate protective RNA interference (RNAi) response. The vcDNA can lead to the host acquiring endogenous viral elements (EVE) in genomic DNA. Thus, we hypothesized that DENV-2 cvcDNA and DENV-2-EVE would arise in C6/36 insect cells challenged with DENV-2 virus in vitro. Here we describe the successful isolation and characterization of cvcDNA constructs homologous to DENV-2 from laboratory challenges with C6/36 cells. At least 1 of these appeared to arise from a DENV-2-EVE. We also show that a cvcDNA preparation derived from the DENV-2 infected in C6/36 insect cells and subjected to rolling circle amplification (RCA) significantly reduced DENV-2 replication when applied to naive C6/36 cells prior to the DENV-2 challenge. This preliminary work lays the groundwork for further studies using the C6/36 cell model to screen and characterize protective EVE against insect and shrimp viruses.

An analysis that combined bioinformatics, comparative sequence/structural analysis, and experimental assays has been completed on respiratory syncytial virus (RSV). Both the genomic RNA and its reverse complement were studied using the novel bioinformatics pipeline ScanFold, which predicted 49 regions on RSV RNAs that appear to encode functional secondary structures (based on their unusually stable sequence order). Multiple motifs appear to be conserved between RSV and related virus strains, including one region within the F gene, which had a highly favorable overall prediction metric of a paired secondary structure. This motif was subjected to additional experimental analyses using SHAPE analysis to confirm ScanFold predicted secondary structure. In subsequent analysis, RSV F mRNA transcripts were made by in vitro transcription using T7 polymerase and transcripts which relaxed the predicted secondary structure yielded slightly higher mRNA transcripts and protein expression levels as wildtype F. However, using reverse genetics for comparison of viruses containing wildtype or relaxed F suggested that the predicted secondary structures may be critical for RSV replication in cells. To our knowledge, this is the first study to examine conserved RNA structures across multiple RSV strains and may help identify potential therapeutic targets to inhibit.

The emergence of new SARS-CoV-2 variants necessitates the reevaluation of current COVID-19 tests to ensure continued accuracy and reliability. The new SARS-CoV-2 variant, Omicron, is heavily mutated, with over 50 mutations within its RNA genome. Any of these mutations could adversely affect the ability of diagnostic assays to detect the virus in patient samples, potentially leading to inconclusive or false negative results. In fact, the U.S. Food and Drug Administration (FDA) has identified over two dozen diagnostic tests that contain a gene target that is expected to have "significantly reduced sensitivity due to a mutation in the SAS-CoV-2 Omicron variant"1. Additionally, one of the U.S. Centers for Disease Control and Prevention (CDC) Emergency Use Authorization (EUA) targets for COVID-19 tests, 2019-nCoV_N1, overlaps an Omicron mutation within the sequence targeted by the fluorescent probe. This target from the CDC has been used in many other EUA assays. Using in vitro transcribed (IVT) N gene RNA representing the wild-type (GenBank/GISAID ID MN908947.3) and Omicron variant (BA.1, GISAID ID EPI_ISL_6752027), we evaluated the performance of two different amplification protocols, both of which include the CDC 2019-nCoV_N1 primer-probe set. Both assays were able to detect the mutant N1 sequence as efficiently as the wild-type sequence. Consequently, these data suggest that diagnostic assays that use the 2019-nCoV-N1 primer-probe set are unlikely to be impacted by currently circulating Omicron lineage viruses.

The 2022 - 2023 highly pathogenic avian influenza (HPAI) virus outbreak of H5N1 Eurasian lineage goose/Guangdong clade 2.3.4.4b is the largest in North American history and has significantly impacted wild bird populations and domestic poultry across the United States. Synanthropic birds may play an important role in transmitting the virus laterally to other wild bird species and domestic poultry. Understanding the prevalence of HPAI H5N1 in different avian orders may help inform management decisions and potential risk factors for both wild and domestic bird populations. Following the confirmation of infection of HPAI H5N1 in domestic poultry at two commercial premises in IN, USA, we sampled and tested 266 synanthropic avian species within the Columbiformes and Passeriformes orders and found no detection of the virus at either location. Additionally, laboratories within the National Animal Health Laboratory Network were queried for influenza Type A rRT-PCR assay test results from morbidity and mortality events in wild birds, consisting of 10,786 birds tested across eight orders and 1,666 avian influenza virus detections. Query results were assessed by taxonomic groups for viral prevalence and suggested that the virus most often was observed in predatory and scavenging birds. Although detections were found in non-predatory synanthropic birds including the orders Columbiformes, Galliformes, and Passeriformes, the risk of transmission from and between these groups appears comparatively low, with apparent prevalence rates of 0.0090, 0.0688, and 0.0147, respectively. The highest prevalence was observed in raptors (0.2514), with prevalence rates in exclusively scavenging Cathartidae reaching up to 0.5333. There is strong evidence that consumption of infected tissues is a key pathway for transmission of avian influenza viruses. Understanding the impact of the 2022 - 2023 HPAI outbreak in wild bird populations can provide pertinent information on viral transmission, disease ecology, and risk to humans and agriculture.

BackgroundChronic hepatitis B virus (HBV) infection remains a major global health burden due to the persistence of covalently closed circular DNA (cccDNA), a stable episomal viral reservoir resistant to current antiviral therapies. The transcriptional activity of cccDNA depends on its chromatin organization, yet the contribution of histone variants to this regulation remains poorly understood. MethodsUsing mass spectrometry-based proteomics on native cccDNA purified from infected primary human hepatocytes, we identified the histone variant H2A.Z and its chaperone SRCAP as cccDNA-associated proteins. Functional analyses combining shRNA-mediated silencing, chromatin immunoprecipitation (ChIP), and ATAC-seq were performed in HBV-infected HepG2-NTCP cells and primary human hepatocytes to characterize their roles in cccDNA formation and transcription. ResultsDepletion of H2A.Z.1, H2A.Z.2, or SRCAP reduced both HBV RNA levels and cccDNA formation. H2A.Z recruitment to cccDNA correlated with the establishment of active chromatin marks (H3K4me3) and enhanced RNA polymerase II loading, promoting an open and transcriptionally active chromatin state. In addition, we identified BRD2, an H2A.Z-associated transcriptional co-activator, as a positive regulator of HBV transcription. shRNA mediated depletion and pharmacological degradation of BRD2 using the PROTAC ARV-771 reduced HBV RNA levels, supporting its potential as an antiviral target. ConclusionOur findings uncover a critical role of the H2A.Z variant and SRCAP complex in cccDNA formation and transcriptional activation. By facilitating chromatin accessibility and RNA polymerase II recruitment, H2A.Z establishes an epigenetic environment favorable to HBV persistence. Targeting H2A.Z-associated co-activators such as BRD2 may represent a promising strategy to silence cccDNA transcription and achieve functional HBV cure.

Australia experienced widespread COVID-19 outbreaks from infection with the SARS-CoV-2 Delta variant between June 2021 and February 2022. Whole-genome sequencing of virus from an early case revealed a sub-consensus level of sequencing reads supporting a 17-nucleotide frameshift-inducing deletion in ORF7a that truncated the peptide sequence. The variant rapidly became represented at the consensus level (Delta-ORF7a{Delta}17del) in most of the outbreak cases in Australia. Retrospective analysis of ORF7a deletions in all GISAID clade GK Delta genomes showed that of 4,018,216 genomes, 134,751 ([~]3.35%) possessed a deletion in ORF7a, with the ORF7a{Delta}17del mutation by far the most common. Approximately 99.05% of Delta-ORF7a{Delta}17del genomes on GISAID originated from the Australian Delta outbreak, and comprised 87% of genomes in the outbreak. In vitro comparison of lineages in cell culture showed a significantly greater proportion of cells were infected with Delta-ORF7a{Delta}17del than with a contemporaneous Delta variant without ORF7a{Delta}17del (Delta-ORF7aintact), and the proportion was also measurably higher than an early SARS-CoV-2 strain (A.2.2). These results showed that Delta-ORF7a{Delta}17del potentially has a slight growth advantage compared to Delta-ORF7aintact. Delta-ORF7a{Delta}17del viruses still produced ORF7a protein, but significantly less than A.2.2, in a different cellular distribution with a more diffuse expression throughout the cytoplasm of infected cells. These data suggest that the proliferation of Delta-ORF7a{Delta}17del genomes during the Australian Delta outbreak was likely not a result of an intrinsic benefit of the ORF7a{Delta}17del mutation, but rather a chance founder effect. Nonetheless, the abundance of different ORF7a deletions in genomes worldwide suggests these have some benefit to virus transmission. IMPORTANCEDeletions in the ORF7a region of SARS-CoV-2 have been noted since early in the COVID-19 pandemic, but are generally reported as transient mutations that are quickly lost in the population. Consequently, ORF7a deletions are considered disadvantageous to the virus through possible loss-of-function effects. In constrast to these earlier reports, we present the first report of a SARS-CoV-2 variant with an ORF7a deletion that dominated for the entirety of a protracted outbreak, and found no associated fitness disadvantage or advantage in cell culture. The relatively common rise and fall of ORF7a deletion variants over time likely represent chance founder events followed by proliferation until a more fit variant(s) is introduced to the population. Our global clade-level survey of ORF7a deletions will be a useful resource for future studies into this gene region.

The order Picornavirales is a group of highly diverse RNA viruses that includes many pathogens of significance to human and veterinary health, agriculture and the wider environment. However, the wide range of viruses assigned to the order, together with their genomic variability, and the recent description of numerous "picorna-like" viruses derived from metagenomic analyses of environmental samples, challenges the existing taxonomic classification of members of the order and the criteria for their classification. Here, we combine the existing gold standard, hallmark RNA-dependent RNA-polymerase (RdRp) gene sequence-based analysis with helicase sequence-based phylogeny, RdRp structural prediction through the use of ColabFold and Fold Tree, and analysis of coding complete genomes using GRAViTy-V2, to genetically classify 525 Picornavirales genomes and recently described "picorna-like" viruses. All analyses were conducted with a bespoke, fully automated pipeline for retrieval of genomes, domain classification and extraction, phylogenetic analysis, and output conditioning, which is available as open-source software. Our results reveal broad support for existing families as well as for six novel families, and 32 new genera. In instances where inconsistencies were found between classification methods, we demonstrate how examination of the pipelines output may be used to reconcile differences with respect to the genomic features quantified by the analysis. Automated multimodal taxonomic analysis may save significant resources over manual methods and better define demarcation criteria for families and genera.

To assess the feasibility of isolating Chlamydia strains with antiviral activity against avian infectious bronchitis virus (IBV) from healthy chickens in farms, 58 Chlamydia strains were obtained from 160 lung tissue samples collected across ten chicken farms, yielding an overall isolation rate of 36.25%. SPF chickens co-infected with Chlamydia and IBV were employed for verification, leading to the identification of eight strains (Bi [&ge;] 0.800) with potent antiviral effects against IBV, accounting for 13.79% of the total isolated strains. The top-performing strain, Y17 Chlamydia strain, was selected and subjected to cell co-culture and U-tube experiments with IBV. Results demonstrated that the Y17 Chlamydia strain significantly impeded IBV replication in chicken tracheal epithelial cells (P<0.01) and did not secrete or induce host cells to secrete extracellular metabolites with antagonistic effects on IBV infection in U-tube experiments (P>0.05), suggesting that its antiviral activity was cell-autonomous. Our research highlights the feasibility of isolating Chlamydia strains with antiviral activity against IBV from healthy chickens and suggests that antiviral strains could be widespread, even though their efficacy against viruses is strain-specific. The presence of broad-spectrum antiviral Chlamydia strains may also be possible. Considering the prevalence of Chlamydia strains in vertebrate hosts, along with the fact that some strains are either non-pathogenic or display low toxicity, our findings could offer a fresh perspective on the prevention and treatment of viral infections in vertebrates.

Short read sequencing of two field-grown plants of red raspberry cv Glen Dee identified sequences derived from Rubus yellow net virus (RYNV). Surprisingly, PCR primers designed to target these sequences amplified RYNV-specific DNA fragments from some high health nuclear stock raspberry plants, previously tested as free from RYNV infection. The complete sequence of a Scottish isolate of RYNV (RYNV LG) was determined and a panel of primers covering the entire virus genome was designed to demonstrate that nuclear stock plants of cultivars Glen Dee, Glen Ericht, Glen Fyne and Glen Moy all contain partial fragments of the RYNV genome as integrated elements but do not contain the entire RYNV genome. The complete, circular genome of RYNV could be detected in RYNV LG-infected plants by rolling circle amplification but was not detected in Glen Dee plants. Grafting experiments showed that infectious RYNV could be transferred from RYNV LG plants but not from Glen Dee or Glen Moy plants, further demonstrating that these cultivars contain only partial, integrated RYNV elements. Grafting of RYNV LG to the certification-approved indexing species R. occidentalis (black raspberry) and R. macraei, showed that RYNV LG induced recognisable symptoms in both plants but that R. macraei was more susceptible and produced more recognizable symptoms. Effective testing for RYNV in high health certified raspberry plants can be achieved by combining graft testing with multifragment PCR.

The family of Geminiviridae consists of more than 500 circular single-stranded (ss) DNA viral species that can infect numerous dicot and monocot plants. Geminiviruses replicate their genome in the nucleus of a plant cell, taking advantage of the hosts DNA replication machinery. For converting their DNA into double-stranded DNA, and subsequent replication, these viruses rely on host DNA polymerases. However, the priming of the very first step of this process, i.e. the conversion of incoming circular ssDNA into a dsDNA molecule, has remained elusive for almost 30 years. In this study, sequencing of melon (Cucumis melo) accession K18 carrying the Tomato leaf curl New Delhi virus (ToLCNDV) recessive resistance quantitative trait locus (QTL) in chromosome 11, and analyses of DNA sequence data from 100 melon genomes, showed a conservation of a shared mutation in the DNA Primase Large subunit (PRiL) of all accessions that exhibited resistance upon a challenge with ToLCNDV. Silencing of (native) Nicotiana benthamiana PriL and subsequent challenging with three different geminiviruses showed a severe reduction in titers of all three viruses, altogether emphasizing an important role of PRiL in geminiviral replication. A model is presented explaining the role of PriL during initiation of geminiviral DNA replication, i.e. as a regulatory subunit of primase that generates an RNA primer at the onset of DNA replication in analogy to DNA Primase-mediated initiation of DNA replication in all living organisms.

Infectious bursal disease virus (IBDV) is endemic worldwide and causes immunosuppression in chickens. We hypothesized that a previous history of IBDV in chickens would render them more susceptible to infection by influenza A viruses (IAVs) from aquatic waterfowl reservoirs. To model this, we inoculated 14 day old specific pathogen free (SPF) chickens with a low pathogenicity avian influenza (LPAI) virus strain from a mallard (A/Mallard/Alberta/156/01 (H3N8)) and compared replication and shedding between immunocompetent chickens and chickens that had immune dysregulation due to a prior IBDV infection with strain F52/70 (genogroup A1B1) at 2 days of age. The mallard IAV strain replicated in the upper respiratory tract of the chickens, and virus was shed from the oropharyngeal cavity, but there was no shedding from the cloaca, and no transmission to sentinel chickens. Replication of the mallard IAV in the chicken host was associated with amino acid substitutions in the polymerase complex and HA. IBDV infection increased the average fold change of IAV replication in the trachea of chickens, prolonged the shedding of infectious IAV from 5 to 6 days in some chickens, increased the number of amino acid substitutions detected in the IAV population from 13 to 30, and significantly increased the number of mutations per IAV sample from 2.50 (SD +/- 1.83) in the Mock/IAV group to 4.75 (SD +/- 1.81) in the IBDV/IAV group (p < 0.01). Taken together, IBDV infection prolonged the shedding of the mallard IAV in some chickens and changed IAV intra-host evolution. Author summarySpillover of IAVs from wild aquatic waterfowl into poultry populations occur frequently, which increases the risk of human infection as people have more contact with poultry than wild birds. Poultry flocks may have other co-morbidities that may influence the spread of IAV. Our data demonstrate that prior IBDV infection increased the average fold change of a mallard H3N8 LPAI virus in the trachea of inoculated chickens, prolonged the shedding of infectious IAV from the oropharyngeal cavity, and significantly increased the average number of amino acid substitutions per IAV sample. We hypothesize that IBDV infection could increase the amount of IAV shed into the environment and broaden the diversity of the IAV population shed. We conclude that controlling the spread of wild aquatic waterfowl strains of IAV in chickens should involve a holistic approach, including the control of co-morbidities and immunosuppressive diseases that could exacerbate their spread.

Infectious Bursal Disease Virus (IBDV) (Avibirnavirus genus, Birnaviridae family) is a non-enveloped virus with a double-stranded RNA (dsRNA) genome. IBDV causes a highly contagious and immunosuppressive disease in domestic chickens (Gallus gallus), representing a major threat to the global poultry industry. Apoptotic cell death and exacerbated innate immune responses have been implicated in IBDV pathogenesis. Previous studies from our laboratory demonstrated the crucial role of type I interferon (IFN) in triggering apoptosis in IBDV-infected cell cultures. Genomic IBDV dsRNA is recognized by the cytoplasmic pattern recognition receptor (PRR) melanoma differentiation-associated gene 5 (MDA5) in chicken cells, triggering type I IFN responses. However, the contribution of the endosomal PRR Toll-like receptor 3 (TLR3) dsRNA sensor on type I IFN production upon IBDV infection has not been studied, despite several studies have demonstrated that its expression is significantly upregulated upon IBDV infection. Here, we demonstrate that ablation of TLR3 gene expression in DF-1 chicken fibroblasts results in a complete blockade of IBDV-induced apoptosis, a marked reduction in IFN production, and a significant enhancement of virus progeny yields. Notably, this effect appears to be specific to IBDV, as similar outcomes were not observed with other RNA viruses. Our findings also suggest that TLR3 may also play a role in viral release into the extracellular space. Additionally, receptor interacting protein kinase 1 (RIPK1), a protein that interacts with TLR3 through the adaptor Toll/IL-1 receptor (TIR) domain-containing adaptor-inducing interferon-{beta} (TRIF), was shown to contribute to both IFN production and apoptosis in response to IBDV infection or dsRNA stimulation in DF-1 cells. Overall, this study provides new insights into the innate immune recognition of IBDV, highlighting the central role of TLR3 in mediating antiviral responses in chicken cells. Author summaryInfectious Bursal Disease Virus (IBDV) is the etiological agent of a devastating syndrome known as IBD or Gumboro disease, which affects domestic chickens (Gallus gallus), leading to significant economic losses in the poultry industry worldwide. The virus primarily targets immature B lymphocytes causing their killing by apoptosis, resulting in severe immunosuppression. Diverse studies have suggested that exacerbation of the innate immune response is related to IBDV pathogenesis. Here, we have investigated the role of the cellular sensor of double-stranded RNA (dsRNA) Toll-like receptor 3 (TLR3) on the fate of IBDV-infected cells. Deletion of the TLR3 gene completely blocks apoptosis induced in IBDV-infected DF-1 cells, drastically reduces interferon (IFN) production and improves viral replication efficiency. We also demonstrated the participation of receptor interacting protein kinase 1 (RIPK1), a known downstream mediator of TLR3, in both, IFN production and apoptosis induction in response to IBDV infection. These findings provide new molecular insights into the mechanisms underlying the robust type I IFN response observed during IBDV infection and contribute to a deeper understanding of IBDV pathogenesis.

Psittacine beak and feather disease virus (PBFDV) is a widespread and highly pathogenic virus in parrots (Psittaciformes), threatening both captive and wild populations over the world. The disease typically presents with feather and beak abnormalities, along with possible immune system suppression. No cure or commercialized vaccine is currently available. Our understanding of the Psittacine beak and feather disease often come from infected individuals with visible symptoms. Limited knowledge exists regarding the pathology and role of asymptomatic individuals in disease transmission. Asymptomatic individuals could shed virus in their crop secretion, feces, or feathers. In this study, we investigated the temporal change in viral load in feather and fecal samples from 17 asymptomatic rosy-faced lovebirds (Agapornis roseicollis). We developed a qPCR assay for PBFDV viral load quantification in the studied lovebirds. Our results showed that most of the individuals had very low viral load, while three individuals with high viral load at the beginning of the experiment were observed to exhibit a decreasing trend in viral load in both fecal and feather samples. Surprisingly, the viral load in an individual can drop from a high level to an undetectable level within three months, which is contrary to the prevailing notion that the disease is highly lethal with few reports of complete recovery. We also showed that viral load in feathers was higher than in feces. Our study provides valuable insights into the infection dynamics of PBFDV in asymptomatic individuals and contribute to the understanding of disease transmission in parrots.

The complete sequence of Wineberry latent virus (WLV), a previously reported but uncharacterised Rubus-infecting virus with flexuous particles, has been determined. Analysis shows WLV to have 76% overall nucleotide sequence identity to the more recently discovered Blackberry virus E, an allexivirus belonging to the subgroup of these viruses that lack the 3 proximal cysteine-rich protein (CRP) gene present in Allium-infecting allexiviruses. An infectious cDNA clone of WLV was constructed and a mutation introduced into the P40 gene, which is an allexivirus-specific gene of unknown function. In addition, the infectious clone was modified to express the green fluorescent protein (GFP) as an N-terminal fusion to the WLV coat protein (CP). Using this GFP overcoat strategy it was possible to follow the multiplication and movement of the virus in infected Chenopodium quinoa and spinach leaves. Introduction of the frameshift mutation into the P40 gene of WLV reduced virus accumulation by 97%, and with the GFP overcoated WLV the P40 mutation almost entirely abolished GFP fluorescence in inoculated leaves suggesting that the WLV P40 protein is required for normal levels of virus multiplication. RepositoriesWLV complete sequences deposited at GenBank. Accession Nos. MZ944847.1, OQ877124.1

In Cote dIvoire, banana (Musa sp.) ranks third among exportation products and represents 3% of the Gross Domestic Product with a national production up to 500000 tons in 2019. Banana is subject to numerous disease agents among which viruses cause significant losses. To figure out the impact of viruses in Ivorian industrial banana fields, surveys were conducted in the 7 main banana production departments. A total of 260 leaf fragments presenting viral symptoms were collected and analyzed. From the 65 leaf fragments used for biological indexing, 14 showed symptoms related to Cucumber mosaic virus (CMV). CMV presence was confirmed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using CMV polyclonal antibodies. CMV strains we isolated, appeared to be highly infectious and to produce various symptoms like mosaic, chlorosis, and necrotic spots on Cucumis sativus, Cucurbita pepo, and Nicotiana tabacum. Satellite RNAs (SatRNAs) associated with CMV isolates were also detected using reverse transcription polymerase chain reaction (RT-PCR) with a degenerate primer pair. CMVs coat protein as well as satRNAs was sequenced. Novel Ivorian coat proteins and satRNAs were compared to publicly available sequences. We noticed a single amino acid substitution (Serine to Leucine) at position 73 of the novel coat protein that allowed us to divide Ivorian CMV strains into two groups. Molecular and phylogenetic analysis suggested that Ivorian strains might be classified into CMV Subgroup IA. We also discovered that satellite RNA associated with Ivorian CMVs form a separate clade.

Avian influenza virus (AIV) epidemiology is well-documented in temperate regions but remains poorly understood in isolated ecosystems like tropical oceanic islands. On these islands, seabirds nest in dense interspecific colonies where the role of different species as reservoirs and dispersers of AIV may vary greatly. Here, we examine the role of noddies (Anous spp.) as potential reservoirs for low pathogenic AIV and evaluate their potential as sentinel species for highly pathogenic AIV introduction on tropical oceanic islands. We analyzed blood samples from 11 seabird species across eight islands in the southwestern Indian Ocean (2015-2020). Noddies exhibited high, stable seroprevalence (30-45%), comparable to reservoir host species in temperate regions. The detection of two N7-positive noddies, sampled the same year on two distinct islands, provided direct molecular evidence that AIV actively circulates on these island colonies. While most other species showed low exposure, Bridled Terns (Onychoprion anaethetus) had exceptionally high seroprevalence (80%), though their reservoir status requires further investigation due to limited sampling. Given noddies consistent exposure and regional distribution, we recommend prioritizing islands with large noddy populations for AIV surveillance. Continued investigation of viral dynamics within and among islands is now called for to elucidate the ecological drivers of AIV maintenance and transmission.

Okra (bhendi) is a widely cultivated food crop in warm regions of the world, with India contributing approximately 60% of global production. However, okra cultivation in India is severely constrained by viral diseases, among which infections caused by the geminiviruses bhendi yellow vein mosaic virus (BYVMV) and okra enation leaf curl virus (OELCuV), in association with their satellites, represent major limitations to crop productivity. In recent years, the geminivirus-encoded C4 protein has emerged as a key pathogenicity determinant in this viral family, with functions that include suppression of multiple layers of plant antiviral defence and induction of disease symptoms. Here, we comparatively characterize the C4 proteins of BYVMV and OELCuV by determining their targeting signals and subcellular localization, and by assessing their ability to induce developmental abnormalities and suppress the cell-to-cell spread of RNA silencing. Our results reveal that the two C4 proteins display distinct subcellular localization patterns, yet both are capable of inducing developmental alterations, likely through different mechanisms, and of suppressing the intercellular spread of RNA silencing, possibly via interaction with a common host factor. Together, these findings suggest that C4 might be a critical virulence factor in okra-infecting geminiviruses and act as a symptom determinant. The C4 proteins encoded by BYVMV and OELCuV therefore emerge as promising targets for the development of antiviral management strategies in okra.

Oseltamivir-resistant influenza viruses arise due to amino-acid mutations in key residues, but these changes often reduce their replicative and transmission fitness. Widespread oseltamivir-resistance has not yet been observed in A(H1N1)pdm09 viruses. However, it is known that permissive mutations in the neuraminidase (NA) of former seasonal A(H1N1) viruses from 2007-2009 buffered the detrimental effect of the NA H275Y mutation, resulting in fit oseltamivir-resistant viruses that circulated widely. This study explored two approaches to predict permissive mutations that may enable a fit H275Y A(H1N1)pdm09 variant to arise. A computational approach used phylogenetic and in silico protein stability analyses to predict potentially permissive mutations, which were then evaluated by in vitro NA enzyme activity and expression analysis, followed by in vitro replication. The second approach involved the generation of a virus library which encompassed all possible individual 2.9 x 104 codon mutations in the NA whilst keeping H275Y fixed. To select for variant viruses with the greatest fitness, the virus library was serially passaged in ferrets (via contact and aerosol transmission) and resultant viruses were deep sequenced. The computational approach predicted three NA permissive mutations, and even though they only offset the in vitro impact of H275Y on NA enzyme expression by 10%, they could restore replication fitness of the H275Y variant in A549 cells. In our experimental approach, a diverse virus library (97% of 8911 possible single amino-acid substitutions were sampled) was successfully transmitted through ferrets, and sequence analysis of resulting virus pools in nasal washes identified three mutations that improved virus transmissibility. Of these, one NA mutation, I188T, has been increasing in frequency since 2017 and is now present in 90% of all circulating A(H1N1)pdm09 viruses. Overall, this study provides valuable insights into the evolution of the influenza NA protein and identified several mutations that may potentially facilitate the emergence of a fit H275Y A(H1N1)pdm09 variant.