Journal of General Virology

Infectious salmon anaemia virus with highly polymorphic region deletions (ISAV-HPR{Delta}) is classified as pathogenic, yet field outbreaks display wide variation in disease severity. To determine the extent of inherent virulence differences among ISAV-HPR{Delta} isolates, we conducted a standardized freshwater bath challenge in Atlantic salmon using ten isolates, including the high-virulent reference strain NO/Glesvaer/2/90 and nine recent Norwegian field isolates. Cumulative mortality, infection kinetics, tissue viral loads, shedding, and pathological changes were characterised through RT-qPCR, histopathology, immunohistochemistry, and flow cytometry. All isolates established systemic infection, but exhibited pronounced differences in infection dynamics, virus shedding, clinical signs, and pathological outcomes. Cumulative mortality ranged from 15% to 100%, allowing separation of isolates into high- ([&ge;]90%), moderate- (40-50%), and low-mortality (<20%) categories. Isolates with high mortality showed rapid systemic spread, extensive endothelial infection, and significant pathology compatible with infectious salmon anaemia. Shedding profiles of virus to water differed substantially and were not clearly correlated with cumulative mortality, viral RNA load in tissues or mortality. High ISAV RNA was detected in water for the H16 isolate with [~]10 - 100-fold higher viral RNA than H20 and [A]. VA and S, although giving high mortality (>90%), had much lower (shedding (highest RNA range 1.1 - 3.6^102). Segment 5 and 6 sequencing confirmed that all isolates carried genetic mutations typical of pathogenic ISAV except [A], that have an atypical mutation in the putative protease cleavage site on segment 5. However, these mutations alone did not account for the wide biological continuum of mortality.

Yellow-eyed penguins (Megadyptes antipodes), or hoiho in te reo M[a]ori, are predicted to become extinct on mainland Aotearoa New Zealand in the next few decades, with infectious disease a significant contributor to their decline. A recent disease phenomenon termed respiratory distress syndrome (RDS) causing lung pathology has been identified in very young chicks. To date, no causative pathogens for RDS have been identified. In 2020 and 2021, the number of chick deaths from suspected RDS increased four- and five-fold, respectively, with a mortality rate of >90%. Here, we aimed to identify possible pathogens responsible for RDS disease impacting yelloweyed penguins. Total RNA was extracted from tissue samples collected during post-mortem of 43 chicks and subject to metatranscriptomic sequencing. From these data we identified a novel and highly abundant gyrovirus in 80% of tissue samples. This virus exhibited only 41% amino acid identity within VP1 to its closest relative, Gyrovirus 8, discovered in a diseased seabird. No other exogenous viral transcripts, nor pathogenic bacterial, protozoal and fungal organisms, were identified in these tissues. Due to the high relative abundance of viral reads, it is likely that this novel gyrovirus is associated with RDS in yellow-eyed penguin chicks. Author SummaryNew Zealands population of yellow-eyed penguins, also called hoiho, are predicted to become extinct in the next 20-30 years, with disease a major factor contributing to their decline. A new disease, causing fluid and bleeding into the lungs, was initially identified in 2019 in very young chicks. It was characterised as causing respiratory distress with a mortality of >90% usually within the first week of life. To date, no causative pathogens of the disease have been identified. We aimed to identify possible pathogens responsible for respiratory disease in these penguin chicks. A metatranscriptomic survey of dead chicks identified a novel and highly abundant gyrovirus present in diseased tissue, with closely related viruses causing disease in other avian hosts. It is, therefore, highly likely that this novel gyrovirus is associated with respiratory disease in these chicks. This finding offers the potential to increase the success of disease management in the critically endangered yellow-eyed penguin and possibly other at-risk penguin species. The potential to lessen mortality and slow the decline of the species is essential in protecting the biodiversity of New Zealands fauna and flora.

Coxsackievirus A9, an enterovirus, is a common cause of paediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to expansion, formation of large pores, externalization of VP1 N-termini and loss of the lipid factor from VP1. Factors such as receptor binding, heat, and acidic pH can trigger capsid expansion in some enteroviruses. Here we show that fatty-acid free bovine serum albumin or neutral endosomal ionic conditions can independently prime CVA9 for expansion and genome release. Our results show that CVA9 treatment with albumin or endosomal ions generates a heterogeneous population of virions, which could be physically separated by asymmetric flow field flow fractionation and computationally by cryo-EM and image processing. We report cryo-EM structures of CVA9 A-particles obtained by albumin or endosomal ion treatment and a control non-expanded virion to 3.5, 3.3 and 2.9 [A] resolutions, respectively. Where albumin promotes stabile expanded virions, the endosomal ionic concentrations induce unstable CVA9 virions which easily disintegrate losing their genome. Loss of most of the VP4 molecules and exposure of negatively-charged amino acid residues in the capsids interior after expansion, create a repulsive viral RNA-capsid interface, aiding genome release. ImportanceCoxsackievirus A9 (CVA9) is a common cause of meningitis and neonatal sepsis. The triggers and mode of action of RNA release into the cell unusually do not require receptor interaction. Rather, a slow process in the endosome, independent of low pH is required. Here, we show by biophysical separation, cryogenic electron microscopy and image reconstruction that albumin and buffers mimicking the endosomal ion composition can separately and together expand and prime CVA9 for uncoating. Furthermore, we show in these expanded particles that VP4 is present at only ~10% of the occupancy found in the virion, VP1 is externalised and the genome is repelled by the negatively-charged, repulsive inner surface of the capsid that occurs due to the expansion. Thus, we can now link observations from cell biology of infection with the physical processes that occur in the capsid to promote genome uncoating.

Immature feathers are known replication sites for high pathogenicity avian influenza viruses (HPAIVs) in poultry. However, it is unclear whether feathers play an active role in viral transmission. This study aims to investigate the contribution of the feather epithelium to the dissemination of clade 2.3.4.4b goose/Guangdong/1996 lineage H5 HPAIVs in the environment, based on natural and experimental infections of domestic ducks. During the 2016-22 outbreaks, H5 HPAIVs exhibited persistent and marked feather epitheliotropism in naturally infected commercial ducks. Infection of feathers resulted in epithelial necrosis, disruption, and the production and release of infectious virions. Viral and feather antigens colocalized in dust samples obtained from poultry barns housing naturally infected birds. In summary, the feather epithelium contributes to viral replication, and it is a likely source of environmental infectious material. This underestimated excretion route could greatly impact the ecology of HPAIVs, facilitating airborne and preening-related infections within a flock, and promoting prolonged viral infectivity and long-distance viral transmission between poultry farms. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/550633v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@16f6b24org.highwire.dtl.DTLVardef@1e33c82org.highwire.dtl.DTLVardef@199b745org.highwire.dtl.DTLVardef@cf2024_HPS_FORMAT_FIGEXP M_FIG C_FIG

Non-enveloped viruses, which lack a lipid envelope, typically display higher resistance to disinfectants, soaps and sanitizers compared to enveloped viruses. The capsids of these viruses are highly stable and symmetric protein shells that resist inactivation by commonly employed virucidal agents. This group of viruses include highly transmissible human pathogens such as Rotavirus, Poliovirus, Foot and Mouth Disease Virus, Norovirus and Adenovirus; thus, devising appropriate strategies for chemical disinfection is essential. We tested a mild combination of a denaturant, alcohol, and organic acid on two representative non-enveloped viruses - Human Adenovirus 5 (HAdV5) and Feline Calicivirus (FCV)- and evaluated the molecular pathway of capsid neutralization using biophysical methods. The transition temperatures signifying conformational shifts in the capsid were established in the presence and absence of chemical treatment using Differential Scanning Calorimetry (DSC), while the corresponding morphological alterations were visualized and correlated using Transmission Electron Microscopy (TEM). We found that while chemical treatment of purified HAdV5 particles resulted in increased thermal instability, followed by large scale particle aggregation; similar treatment of FCV particles resulted in complete collapse of the capsids. The distinct effects of the chemical treatment on the morphology of HAdV5 and FCV suggests that non-enveloped viruses with icosahedral geometry can follow different molecular pathways to inactivation. Further, while individual components of the chemical formulation caused significant damage to the capsids, a synergistic action of the whole formulation was evident against both non-enveloped viruses tested. Molecular level understanding of inactivation pathways may result in the design and development of effective mass-market formulations for rapid neutralization of non-enveloped viruses. HighlightsO_LIformulation consisting of 3.2% citric acid, 1% urea in 70% ethanol, pH4 effectively inactivates HAdV5 and FCV. C_LIO_LIinactivation pathways with complete formulation, are different for the two viruses. C_LIO_LIeffect of whole formulation is more effective compared to individual components. C_LI

New variants of SARS-CoV-2 are continuing to emerge and dominate the regional and global sequence landscapes. Several variants have been labelled as Variants of Concern (VOCs) because of perceptions or evidence that these may have a transmission advantage, increased risk of morbidly and/or mortality or immune evasion in the context of prior infection or vaccination. Placing the VOCs in context and also the underlying variability of SARS-CoV-2 is essential in understanding virus evolution and selection pressures. Sequences of SARS-CoV-2 in nasopharyngeal swabs from hospitalised patients in the UK were determined and virus isolated. The data indicated the virus existed as a population with a consensus level and non-synonymous changes at a minor variant. For example, viruses containing the nsp12 P323L variation from the Wuhan reference sequence, contained minor variants at the position including P and F and other amino acids. These populations were generally preserved when isolates were amplified in cell culture. In order to place VOCs B.1.1.7 (the UK Kent variant) and B.1.351 (the South African variant) in context their growth was compared to a spread of other clinical isolates. The data indicated that the growth in cell culture of the B.1.1.7 VOC was no different from other variants, suggesting that its apparent transmission advantage was not down to replicating more quickly. Growth of B.1.351 was towards the higher end of the variants. Overall, the study suggested that studying the biology of SARS-CoV-2 is complicated by population dynamics and that these need to be considered with new variants. ImportanceSARS-CoV-2 is the causative agent of COVID-19. The virus has spread across the planet causing a global pandemic. In common with other coronaviruses, SARS-CoV-2 genetic material (genomes) can become quite diverse as a consequence of replicating inside cells. This has given rise to multiple variants from the original virus that infected humans. These variants may have different properties and in the context of a widespread vaccination program may render vaccines less ineffective. Our research confirms the degree of genetic diversity of SARS-CoV-2 in patients. By isolating viruses from these patients, we show that there is a 100-fold range in growth of even normal variants. Interestingly, by comparing this to the pattern seen with two Variants of Concern (UK and South African variants), we show that at least in cells the ability of the B.1.1.7 variant to grow is not substantially different to many of the previous variants.

Human adenoviruses (HAdV) are widespread pathogens causing infections of the respiratory and gastrointestinal tracts, genitourinary system and the eye. Species D (HAdV-D) are the most diverse species and cause both gastrointestinal tract infections and epidemic keratoconjunctivitis (EKC). Despite being significant pathogens, HAdV-D are understudied and knowledge around basic mechanisms of cell infection is lacking. Sialic acid (SA) usage has been proposed as a major mechanism of cell infection for EKC causing HAdV-D. Here, we provide apo state crystal structures for fiber knob proteins of 7 previously undetermined HAdV-D, and provide crystal structures of HAdV-D25, HAdV-D29 and HAdV-D53 knob proteins bound to SA. Biologically, we demonstrate that removal of cell surface SA reduced infectivity of HAdV-C5 vectors pseudotyped with HAdV-D fiber knob proteins, whilst engagement of the classical HAdV receptor, CAR was variable. Together, these data indicate an important role for SA engagement in the tropism of many HAdV-D and may facilitate the development of suitable antivirals to control EKC outbreaks.

Influenza A viruses (IAV) are clinically important pathogens that cause seasonal epidemics and pandemics in humans. IAV produce pleomorphic, enveloped virions, which can range from a spherical or bacilliform morphology, the predominant form in the most commonly studied laboratory strains, to long filamentous virions which are characteristic of clinical and veterinary isolates. Understanding the structure and function of filamentous virions is crucial for clarifying their role in viral persistence and immune evasion, and for informing the development of therapeutics that target their entry and/or egress pathways. Structural characterisation of influenza virions is challenging however owing to their fragility, heterogeneity and compared to most virus particles, unusually large size. Here, we combined structural and compositional approaches with integrative modelling to define the complete molecular architecture of influenza virions. In doing so we provide the first description of distinctive structural features of IAV filaments, including the selective incorporation of lipids, specific enrichment of viral and host proteins, and a viral cytoskeleton including a secondary helical layer within the viral capsid and extended fibrils of cofilactin. Collectively our findings suggest an important regulatory role for cofilactin in driving filament morphogenesis and provide important insights into the organisation and composition of IAV filamentous virions.

The 2020/2021 epidemic in Europe of highly pathogenic avian influenza virus (HPAIV) of subtype H5 surpassed all previously recorded European outbreaks in size, genotype constellations and reassortment frequency and continued into 2022 and 2023. The causative 2.3.4.4b viral lineage proved to be highly proficient with respect to reassortment with cocirculating low pathogenic AIV and seems to establish an endemic status in northern Europe. A specific HPAIV reassortant of the subtype H5N3 was detected almost exclusively in red knots (Calidris canutus islandica) in December 2020. It caused systemic and rapidly fatal disease leading to a singular and self-limiting mass mortality affecting about 3.500 birds in the German Wadden Sea, roughly 1% of the entire flyway population of islandica red knots. Phylogenetic analyses revealed that the H5N3 reassortant very likely had formed in red knots and remained confined to this species. While mechanisms of virus circulation in potential reservoir species, dynamics of spill-over and reassortment events and the roles of environmental virus sources remain to be identified, the year-round infection pressure poses severe threats to endangered avian species, and prompts adaptation of habitat and species conservation practices. One-Sentence SummaryHigh red knot mortality in Europe (December 2020) was associated with infection of a unique genotype of HPAIV H5N3 clade 2.3.4.4b.

Withdrawal statementThe authors have withdrawn their manuscript because since deposition of this preprint, they have become aware of the possibility that the findings reported may be an artefact. Carter et al. (J Struct Biol 2018 Jan;201(1):15-25. doi: 10.1016/j.jsb.2017.10.009) have reported that multilamellar bodies exhibit autofluorescence under cryogenic conditions. Thus, there is a possibility that stress induced by virus infection may stimulate formation of the features reported in this article, and autofluorescence, rather than specific labelling led them to identify the puncta as compartments containing RSV proteins or genomes. They are working to address these issues. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

Between 2013-2017, A/Anhui/1/13-lineage (H7N9) low pathogenicity avian influenza virus (LPAIV) was epizootic in chickens in China causing mild disease, with 616 fatal human cases. Despite poultry vaccination, H7N9 has not been eradicated. Previously we demonstrated increased pathogenesis in turkeys infected with H7N9, correlating with the emergence of the L217Q (L226Q H3 numbering) polymorphism in the haemagglutinin (HA) protein. A Q217 containing virus also arose and is now dominant in China following vaccination. We compared infection and transmission of this Q217 containing turkey-adapted (ty-ad) isolate alongside the H7N9 (L217) wild-type (wt) virus in different poultry species, and investigated the zoonotic potential in the ferret model. Both wt and ty-ad viruses demonstrated similar shedding and transmission in turkeys and chickens. However, the ty-ad virus was significantly more pathogenic than the wt virus in turkeys but not in chickens, causing 100% and 33% mortality in turkeys respectively. Expanded tissue tropism was seen for the ty-ad virus in turkeys but not chickens, yet the viral cell receptor distribution was broadly similar in visceral organs of both species. The ty-ad virus required exogenous trypsin for in vitro replication yet had increased replication in primary avian cells. Replication was comparable in mammalian cells and the ty-ad virus replicated successfully in ferrets. The L217Q polymorphism also affected antigenicity. Therefore, H7N9 infection in turkeys can generate novel variants with increased risk through altered pathogenicity and potential HA antigenic escape. These findings emphasise the requirement for enhanced surveillance and understanding of A/Anhui/1/13-lineage viruses and their risk to different species.

Mosquito-borne viruses are emerging in new regions with increasing regularity. Shifting climatic envelopes, especially in temperate regions, and altered enzootic cycles are changing likelihoods of persistence, meaning new approaches are required to predict and monitor establishment. Usutu virus (USUV) is a mosquito-borne viral zoonosis, which was first recorded in the United Kingdom in wild birds and mosquitoes in 2020. By developing a multidisciplinary approach, incorporating enhanced passive and active surveillance of wild birds, we discovered previously unrecorded introductions and identified USUV persistence and geographic expansion. By combining molecular and serological testing with citizen science derived datasets, we show an associated marked decline in a common, widespread, and highly susceptible host species (Eurasian Blackbird, Turdus merula). However, the clearest continuous signal of host population decline remains localised to a large urban area, despite the much wider virus distribution. This may be a consequence of an urban heat island effect and associated increase in length of the mosquito active season. Our results indicate that the establishment and impact of future mosquito-borne pathogens in temperate zones, anticipated under climate change, may first be most apparent in urban areas. Consequently, enhanced surveillance efforts in urban areas could provide an efficient mechanism for detecting novel pathogen emergence. The epidemiology of emerging vector-borne diseases, especially in temperate areas, depends on the interplay between climate, land-use and available hosts; therefore, considering each in isolation will not be sufficient to reliably inform surveillance or response planning.

Zika virus (Orthoflavivirus zikaese) is part of the Orthoflavivirus genus that contains several viral species posing major and expanding burdens to public health worldwide. Upon infection, orthoflaviviruses produce viral RNA fragments named sfRNAs (short flaviviral RNA), generated from viral genomic RNA incompletely degraded by Xrn1/Pcm, the major cellular 5-3 exonuclease. Several conserved elements in the orthoflavivirus 3 untranslated region (UTR) are responsible for XrnI-resistance, named xrRNA. The ubiquitous prevalence of xrRNA/sfRNA in flaviviruses indicate an important role of these structures in the viral life cycle, although their function is not completely understood. Here, we used in vitro reconstitution and infection models to examine the role of ZIKV xrRNA2 in mammalian and insect cells. SHAPE RNA structure probing revealed that disruption of ZIKV xrRNA2 does not affect xrRNA1 or dumbbell structure elsewhere in the 3UTR despite disrupting all sfRNA accumulation in mammalian cells. In vitro RNA degradation assays showed that xrRNA1 efficiently stalled human or mosquito Xrn1 independent of xrRNA2 integrity. Reversion in mammalian cells occurred at early passages in protein kinase R (PKR)-/- or IFN-receptor1 (IFAR1)-/-/PKR-/- knock out (KO) or double KO mammalian A549 cells, respectively, while wild-type virus outcompeted the xrRNA2 defective virus in RNAi-deficient mosquito cells. Together, our data reveals novel details of the mechanism of sfRNA accumulation in mammalian and insect cells We demonstrate a differential sensitivity to the absence of a functional xrRNA2 structure in mammalian and insect cells, with reversion to an active xrRNA2 occurring more rapidly in mammalian cells. Together, our work reveals new details on the requirements for ZIKV sfRNA accumulation in mammalian and mosquito cells.

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 effect of heat on SARS-CoV-2/England/2/2020 viability was assessed by plaque assay and virus culture. Heating to 56{degrees}C and 60{degrees}C for 15, 30 and 60 minutes led to a reduction in titre of between 2.1 and 4.9 log10 pfu/ml but complete inactivation was not observed. At 80{degrees}C plaques were observed after 15 and 30 minutes of heating, however after 60 minutes viable virus was only detected following virus culture. Heating to 80{degrees}C for 90 minutes and 95{degrees}C for 1 and 5 minutes resulted in no viable virus being detected. At 56{degrees}C and 60{degrees}C significant variability between replicates was observed and the titre often increased with heat-treatment time. Nucleic acids were extracted and tested by RT-PCR. Sensitivity of the RT-PCR was not compromised by heating to 56{degrees}C and 60{degrees}C. Heating to 80{degrees}C for 30 minutes or more and 95{degrees}C for 1 or 5 minutes however, resulted in an increase of at least three Ct values. This increase remained constant when different dilutions of virus underwent heat treatment. This indicates that high temperature heat inactivation of clinical samples prior to nucleic acid extraction could significantly affect the ability to detect virus in clinical samples from patients with lower viral loads by RT-PCR.

The ongoing outbreak of Marburg virus disease (MVD) in Rwanda marks the third largest historically, though it has exhibited the lowest fatality rate. Genomic analysis has identified a lineage with limited internal diversity most closely related to a genome sequence from a sporadic case sampled in 2014 in Uganda, though the lineages have diverged from a common ancestor that was circulating for decades in the animal reservoir. Notably, the data also provide evidence that the outbreak resulted from a single zoonotic transmission event with limited human-to-human transmission, rather than multiple independent zoonotic transmission events. The Rwandan MVD outbreak prompted a thorough investigation that included contact tracing, clinical assessment, travel history, sequencing, and serology testing, to trace the viruss origin. Results of investigations linked the index case to a mining cave inhabited by Rousettus aegyptiacus (the Egyptian fruit bat), where three individuals tested seropositive for IgG and IgM, further supporting the zoonotic origin of the outbreak through human-animal interactions.

Viruses are numerically the most abundant forms on Earth, and most are present in soil. Even though viruses are highly abundant in soil and critical to rhizosphere function, visualizing the diverse morphotypes within soil has been challenging. The difficulty is primarily due to the heterogenous nature of isolated suspensions that typically contain nanometer to micron scale debris which renders protein crystallography for structural studies unfeasible and hinders cryo-electron microscopy due to ice thickness and contrast issues. Here we employed and compared a simple spin filtration method to cleanup solutions of extracted viruses for direct observation with cryo-electron microscopy. Although relatively simple, the method employs common physical biochemical separation steps to remove large and small debris which dramatically improves image quality and preservation of structural features to permit visualizing morphotypes not typically seen with conventional negative stain approaches. In addition to tailed and non-tailed polyhedral phages, several under reported or novel morphotypes of soil viruses are directly visualized as a particle library with both 2D and 3D information.

The internationally recognised method for diagnosis of avian influenza (AI) is virus isolation (VI) in specific pathogen-free embryonated fowls eggs (EFEs). In Great Britain (GB), AI virus isolation currently involves two passages in EFEs; the first typically of two days duration followed by a second lasting up to four days meaning that premises may remain under restriction for up to six days. Shorter time lengths for AIV isolation were investigated to reduce the time that businesses remain under official restrictions to safely negate AI infection, whilst maintaining test sensitivity. Both experimental inoculations of EFEs and analyses of VI attempts from high pathogenicity (HP) AI disease incursions in GB since 2016 demonstrated that HP viruses were isolated during first passage while for low pathogenicity AI outbreaks, the second passage could be reduced to two days. Power analysis showed that the benefit of reducing the number of days outweighed the risk of missing a positive isolate. This approach will substantially reduce costs to government and industry by releasing restrictions at least two days earlier where samples are negative for viral nucleic acid. Critically, it will reduce welfare implications of housing birds under restriction and improve international standards without loss of test performance.

Seaweed derived compounds are a renewable resource utilised in the manufacturing and food industry. This study focuses on an Enriched seaweed extract (ESE) isolated from Ascophyllum nodosum. ESE was screened for antiviral activity by plaque reduction assays against influenza A viruses (IAV) H1N1 and H3N2 subtypes. Time of addition assays and FACS analysis were used to help determine the modes of action. The therapeutic potential of ESE was then explored using differentiated human bronchiole epithelial cells at the air liquid interphase and a murine model challenged with IAV. The data indicates ESE primarily interacts directly with virions, preventing virus cell binding. Interestingly, ESE also inhibits early and late stage of the influenza A lifecycle when treatment occurs after cell binding. This inhibitory effect appears to prevent internalisation of virus and release of progeny virus by targeting neuraminidase activity. Intranasal administration of ESE in mice infected with IAV reduced viral load in lung tissue. ESE may be a promising broad acting antiviral agent in the treatment of influenza infections.

The Sarthroviridae are a family of highly compact satellite RNA viruses comprising one recognised species, extra small virus (XSV). Macrobrachium rosenbergii nodavirus (MrNV) is the associated helper virus of XSV and their co-infection has been linked to white tail disease in freshwater prawns globally, although the role of XSV is remains unclear. Here, we describe the discovery and characterisation of ten novel, highly divergent sarthrovirus species from a range of hosts and environments within a small geographical region in Australia. These comprise novel sarthroviruses associated with marine sponges, seal and dingo faeces, environmental marine sediment samples and Indo-Pacific geckos (Hemidactylus garnotii). All the novel viruses possess only a capsid protein, consistent with the genome of XSV, yet exhibit substantial sequence divergence. Notably, some sarthrovirus variants seem to utilise different replication systems despite being genetically identical and present in the same host species. Sequences from nodaviruses, which could plausibly act as helpers, were associated with some, but not all, the sarthroviruses identified here. Phylogenetic analyses support the expansion of the Sarthroviridae into multiple distinct lineages, comprising at least seven genera. Collectively, these findings reveal a broader ecological distribution and evolutionary diversity of sarthroviruses and highlight the possibility of alternative replication strategies and tissue tropism in diverse animal host. SignificanceSarthoviruses are small ([~]800 nucleotides) satellite RNA viruses associated with a nodavirus of crustaceans that acts as a helper. To date, the only known sarthovirus is extra small virus (XSV), which also represents the sole species within the Sarthroviridae. Here, we report the detection of ten divergent sarthroviruses sampled from diverse animal hosts, including vertebrates, that expand the family to 11 species and at least seven genera. These viruses were detected from various host taxa and environmental samples from a confined geographical region in eastern Australia, suggesting that they are ecologically connected. Notably, we did not detect nodaviruses in all samples containing sarthroviruses, suggesting that different viruses may act as helpers for sarthovirus replication.