Flaviviridae. These viruses closely related to flaviviruses are unique due to the segmented nature of their genome. The prototype jingmenvirus, Jingmen tick virus (JMTV), was discovered in Rhipicephalus microplus ticks collected from China in 2010. Jingmenviruses genomes are composed of four to five segments, encoding for up to seven structural proteins and two non-structural proteins, both of which display strong similarities with flaviviral non-structural proteins (NS2B/NS3 and NS5). Jingmenviruses are currently separated into two phylogenetic clades. One clade includes tick- and vertebrate-associated jingmenviruses, which have been detected in ticks and mosquitoes, as well as in humans, cattle, monkeys, bats, rodents, sheep, and tortoises. In addition to these molecular and serological detections, over a hundred human patients tested positive for jingmenviruses after developing febrile illness and flu-like symptoms in China and Serbia. The second phylogenetic clade includes insect-associated jingmenvirus sequences, which have been detected in a wide range of insect species, as well as in crustaceans, plants, and fungi. In addition to being found in various types of hosts, jingmenviruses are endemic, as they have been detected in a wide range of environments, all over the world. Taken together, all of these elements show that jingmenviruses correspond exactly to the definition of emerging viruses at risk of causing a pandemic, since they are already endemic, have a close association with arthropods, are found in animals in close contact with humans, and have caused sporadic cases of febrile illness in multiple patients. Despite these arguments, the vast majority of published data is from metagenomics studies and many aspects of jingmenvirus replication remain to be elucidated, such as their tropism, cycle of transmission, structure, and mechanisms of replication and restriction or epidemiology. It is therefore crucial to prioritize jingmenvirus research in the years to come, to be prepared for their emergence as human or veterinary pathogens.Jingmenviruses are a group of viruses identified recently, in 2014, and currently classified by the International Committee on Taxonomy of Viruses as unclassified  Flavivirus, family Flaviviridae, includes 53 officially identified species and 38 related unclassified viruses   . Flaviviviruses) . Exampleviruses) . Most ofviruses) . Howeverviruses) .The genome of flaviviruses is composed of an 11 kilobase-long single molecule of positive single-stranded RNA (+ssRNA), with no polyadenylation at the 3 prime end, and is encapsidated in an enveloped ∼45 nm icosahedral virus particle . The genRhipicephalus microplus ticks collected in the Jingmen region of Hubei province in China in 2010 , prior to the characterization of JMTV , Brazil (25–67%), Trinidad and Tobago (6–46%), and the French Antilles (24–77%) over the years , Europe , the Americas  and Africa  see . To datehe years . Howeverd Ixodes , as welld Ixodes . The locectively . While ttralasia . The dise change . An examontinent .Apodemus uralensis (29%) and Microtus arvalis (24%)  .In concordance with RNA detection, anti-JMTV antibodies have been found in cattle from three regions of China with 18–37% seroprevalence . MoreoveTo date, only limited ecological data relate to the transmission cycle of this virus. Indeed, JMTV was detected in adult ticks, both males and females, as well as in nymphs and unfed larvae, suggesting it could be vertically transmitted . It has R. microplus, tick), C6/36 cells , DH82 cells  or Vero E6 cells  at low titers and did not necessarily produce cytopathic effect (CPE) (R. microplus) cell line with a 3-log titer increase by day 7, but the number of positive passages is not mentioned. These limited models enabled the observation of thin sections of infected DH-82 cells and their negatively stained supernatant by transmission electron microscopy which suggested that JMTV particles are enveloped and slightly larger than flaviviruses, with a diameter of 70–80 nm and clear protrusions , LLC-PK1 , PK-15 , MDBK , Vero , HEK-293 , DF-1 , and new-born mice  . Jia et trusions . Other, orn mice . Interes culture . SettingJingmenvirus.Since 2014, segmented flavi-like virus sequences have been detected worldwide. Some of these sequences share over 95% identity with JMTV  and are therefore likely to all belong to the same species . SequencJingmenvirus genus (A novel jingmenvirus tentatively named Alongshan virus (ALSV) was isolated and sequenced from a blood sample taken from a human patient reporting tick bites in China in 2017 . The genus genus .I. persulcatus, I. ricinus, D. nuttalli, D. reticulatus, Hae. concinna, Hae. longicornis, Anopheles yatsushiroensis, Ae. vexans, Cx. pipiens pallens, and Cx. tritaeniorhynchus, as well as from sheep, cattle, and deer sera, from locations in Eurasia: in China, Finland, Russia, Serbia, Germany, and France  can successfully sustain persistent infection by a Russian strain of ALSV, over up to several years, with no CPE. Balb/c mice were inoculated intraperitoneally with cell culture supernatant containing a Chinese strain of virus, with 107 copies of RNA and were found to present pathological changes in the liver, kidneys, and brain 14 days post-injection (7 RNA copies/mL), as well as in the kidney and heart tissues (106 RNA copies/mL) but to a lesser extent in brain tissues and blood (104 RNA copies/mL) 30 days post-infection , or in U-87MG, HFF, Caco-2, SK-N-SH, and CRL-2088  cell lines . Such post-translational modifications may shield the virus from recognition by the host’s immune system  . The ATPD. nuttalli and 18 from unspecified tick species. YGTV RNA was subsequently detected in two D. marginatus and one D. nuttalli samples during a tick survey in Russia during the period 2011-2019. Although the number of samples remains low, YGTV seems to be associated to Dermacentor spp. ticks  encodes the complete putative glycoprotein, which shares approximately 62% homology to JMTV, ALSV, and YGTV (from BLAST results). A MUSCLE alignment using the available glycoprotein sequences of tick- and vertebrate-associated jingmenviruses shows that this sequence shares between 51 and 55% homology with one group of tick- and vertebrate-associated jingmenviruses  and between 27 and 30% with the other group . This virus could not be included in our phylogenies  was identified in a metagenomics study of ticks from New South Wales in Australia . The seqlogenies  since noFinally, Xinjiang tick virus 1 was detected from unspecified ticks from China [unpublished data, deposited on Genbank by Pteropus lylei bats from Cambodia  collected from Trinidad, Peru, and Panama between 2008 and 2013, and subsequently from two Culex spp. mosquito pools collected from Brazil in 2010 , which was isolated from six  in 2010 ; Table 2 in 2010 . Segment in 2010 . Zhang e in 2010 .Ae. albopictus, CT Cx. tarsalis, Aag2 Ae. aegypti) and intrathoracically inoculated female mosquitoes , but not in tick (ISE6 I. scapularis), sand-fly  or vertebrate  cell lines, or in intracranially inoculated new-born mice. No significant vertical transmission was detected in mosquitoes. Viral replication was detected in three mosquito cell lines (C6/36 Culex-associated jingmenvirus tentatively named Mole Culex virus (MoCV) was isolated from three pools of Culex mosquitoes collected from Ghana in 2016 and caused CPE on C6/36 cells . Of note is the fact that human patients were found to be seropositive against recombinant proteins from the putative structural ORFs  from France, Germany, and Austria   . All of , adult) . StudyinThe discovery of segmented flavi-like viruses was a real breakthrough in virus research as they were the first examples of a segmented genome at least partly derived from an unsegmented genome . The evoin vitro and in vivo. The influence of this characteristic on the host restriction displayed by others jingmenviruses in laboratory models should be evaluated. The fact that GCXV branches within the insect-associated jingmenvirus subgroup suggests that it shares a common ancestor with a number of other jingmenviruses. This could suggest that many other jingmenviruses are multicomponent, but this remains to be formally demonstrated. The current knowledge on the structural organization of jingmenviruses is somewhat contradictory, since JMTV was measured to be 70–80 nm, ALSV was found to be 80–100 nm for the Chinese strain and 40 nm with 13 nm smaller particles for the Russian strain, and GCVX was measured at 30–35 nm (The discovery of GCXV and its characterization as a multicomponent flavi-like virus was a second breakthrough for jingmenvirus-associated virus research since it was the first report of a multicomponent viral system in an animal virus . Indeed,30–35 nm . The reaIn conclusion, jingmenvirus research should be prioritized research topic as they have the potential to emerge as human or veterinary pathogens. Jingmenviruses are found worldwide, including in tick species that are widespread, in close contact with vertebrates, and known to be vectors for other diseases. They can be associated with ticks and vertebrates and could follow a vector-borne transmission cycle. Tick-associated jingmenviruses have been associated with febrile illness in humans. Finally, they are under-studied and therefore the scientific and clinical communities need better preparedness to face a potential future epidemic.AC, BC, and RC: conceptualization and writing – review and editing. AC: investigation and writing – original draft. BC and RC: resources. AC and BC: visualization. All authors contributed to the article and approved the submitted version.


Aloe vera, curcumin, vitamin C-enriched natural antioxidant, and anti-inflammatory biomolecules are potential ways to prevent or delay ageing. Eggshell membrane (ESM) is also a rich source of collagen; glycosaminoglycans (GAGs) also play an essential role in healing and preventing ageing. It is important to use an extended therapeutic process to prolong the effectiveness of these products, despite the fact that they all have significant anti-ageing properties and the ability to regenerate healthy cells. Encapsulated herbal components are therefore designed to overcome the challenge of ensuring continued treatment over time to prolong the effects of a bioactive component after in situ administration. To study their synergistic effects on a cellular level, alginate, Aloe vera, and orange peel extract were encapsulated in bio-polymeric foaming beads and modified with eggshell membrane protein (ESMP) at various concentrations : . Analysis of the structural and functional properties of foaming beads showed interconnected 3D porous structure, a surface-functionalized group for entrapment of ESMP, and a significant reduction in pore size (51–35 m) and porosity (80%–60%). By performing DPPH assays, HRBC stabilization assays, and antibacterial tests, the beads were assessed as a natural anti-ageing product with sustained release of molecules effective against inflammatory response, oxidative stress, and microbial contamination. MTT assays were conducted using in vitro cell cultures to demonstrate cytocompatibility (in mouse 3T3 fibroblast cells) and cytotoxicity (in human carcinoma HeLa cells). Our study demonstrates that bio-polymeric ESMP beads up to 2 g (A-Av-OP-ESMP2) are practical and feasible natural remedies for suspending defective cell pathways, preventing cell ageing, and promoting healthy cell growth, resulting in a viable and practical natural remedy or therapeutic system.Several therapies and cosmetics are available commercially to prevent or delay cell ageing, which manifests as premature cell death and skin dullness. Use of herbal products such as  In the current scenario, biogerontology is a primary area of focus for researchers and scientists . The ageCosmetic and therapeutic products, such as anti-ageing creams, masks, sunscreen lotions, moisturizers, and several other anti-ageing medicines, have been associated with prolonged side effects and poor outcomes . Herbs ovia physiochemical characterization and in vitro testing for applicability as an anti-ageing product.Anti-ageing products contain chemicals from natural products, such as α-hydroxy acids, salicylic acid, hyaluronic acid, and ascorbic acid, along with preservatives for long-term shelf-life . In addiIt was reported that the lubricant aspect of Aloe vera, enriched with vitamins and fibres, has antioxidant and hydrating gel properties that protect the skin from radiation and thermal or solar burn. It has a prophylactic effect on damaged skin tissue through natural hydration and an antibiotic effect that leads to soothing, cooling, increased elasticity, and promotion of the youthful properties of skin. Clinically, different products are approved to treat skin problems, promote wound healing, slow ageing, heal scratches, and as cleansers to purge the body or skin of impurities .Another essential component of healthy skin is hyaluronan or hyaluronic acid. It is a significant component of ESM and has the potential to retain water molecules by efficiently binding to them, thereby maintaining the moisture content of the cell . AdditioCitrus fruits are rich sources of vitamin C, and α-hydroxy acid has natural antibacterial and antioxidant properties. They play vital roles in deferring ageing by enhancing the synthesis of collagen to improve the elasticity and flexibility of the dermal layer of the skin . ProductAloe vera, ESM hydrolysate, and curcumin; these natural matrices for the delivery of therapeutic products for wound dressing and tissue regeneration have demonstrated significant benefit , calcium chloride, and acetic acid were purchased from HI media Laboratories, India. All the reagents/chemicals used for fabricating hydrogel beads and in their characterization were of cell culture grade.Raw material for fabricating bio-polymeric beads was collected, including raw eggshell, orange peel from the campus canteen, and fresh (a) ESMP preparation: ESMP solution was prepared according to the previously published protocol , which i(b) Preparation of orange peel powder: Collected orange peel was thoroughly washed in ddH2O and dried in a hot-air oven at 37°C. After drying, a fine powder was prepared using a food grinder. The powder was stored in air-tight plastic tubes.(c) Extraction of Aloe vera gel: Fresh Aloe vera leaves were collected from the college herbal garden. The leaves were washed thoroughly with ddH2O, and the top and bottom layers of the leaves were peeled off. The thick, clear gel, or pulpy content of the leaves was collected at the centre. Subsequently, the pulp was blended in a grinder to produce a clear Aloe vera extract.via simple drop-extrusion methods (21G syringe at a height of 10 cm), using a solution of CaCl2 (1 wt %) as a crosslinking agent and ESMP at different ratios, as indicated in (d) Preparation of gel beads: Bio-polymeric beads were prepared following the previously published protocol, with some modification in bead composition . We geneBio-polymeric herbal beads of alginate, Aloe vera, and orange peel extract with entrapped ESMP were synthesized using the drop-extrusion crosslinking method in calcium chloride solution. Subsequently, characterization of fabricated samples was carried out to evaluate their applicability in anti-ageing and cell regeneration processes.2. Lastly, the beads were filtered and separated from the solution, then freeze-dried for prolonged storage.Fabricated beads were kept in crosslinking solution for 5 min to facilitate efficient crosslinking and ESMP entrapment, then gently washed with ddH2O to remove unbound particles and excess CaClThe macro-structure of the bio-polymeric beads was determined visibly by macroscopic examination and size dimension (diameter in mm) measurement of a randomly selected 50 beads from each sample using a vernier caliper. However, ultra-structure of the beads was analyzed by studying the cross-sectional area of images of five randomly collected beads captured using a field emission scanning electron microscope . The pore size and interconnectivity of the beads were determined by analyzing microscopic images using ImageJ software, and the average dimensions of the beads were determined.−1 at 2 cm−1 resolution was assessed using an FTIR spectrophotometer , and the generated spectra were analyzed.The functional and intermolecular interaction that occurs between the samples was determined by FTIR spectra analysis. All the samples were prepared following a previously published protocol . Brieflyn = 5):1); then the volume of solvent after immersion of the beads, V2, was measured. The volume, V3, was also measured after removal of the beads at regular time intervals. Vt represents the total volume.Good porosity is required for absorption, vascularization, transport, and release of drug molecules. Therefore, we measured the porosity (χ) of the beads using the liquid dispersion method . We meass is the weight of wet beads and Wd is the weight of dried beads.Similarly, bead hydrophilicity, or swelling ratio (S), was determined by immersing the beads in 1X PBS solution for 1 h at room temperature (RT) and measuring their weight at a regular time interval of 10 min. The following equation is used to measure S . Here, Wn = 5), and the mean value was calculated.All the experiments were repeated five times (n = 3) was calculated using 0 denotes the initial weight of the scaffold and Wt denotes the weight of the degraded scaffold at different time intervals.The rate of degradation of beads was used to measure their stability and was determined under non-enzymatic conditions in a PBS buffer system . BrieflyThe release of bioactive components from the polymeric bead surface after incubation in PBS buffer was measured using the total immersion method. The absorbance of the ESMP encapsulated bead samples was recorded using a UV-Vis spectrophotometer, and the calibration curve of ESMP (50 μg/ml) was prepared for monitoring drug release. The modified beads were immersed in 150 ml of 1X PBS and placed in a shaking water bath at 37°C. After 12 h of incubation, 2 ml of the sample were taken out at regular time intervals of 30 min, and the absorbance at 412 nm was recorded. After measuring absorbance, the sample was reintroduced into the main flask to maintain constant final volume. The drug release percentage was obtained by comparison with the standard calibration curve.In vitro antibacterial activity of the beads was tested against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria, as described previously , of either 3T3 mouse fibroblast cells or HeLa carcinoma cells, were seeded over the beads  in the tissue culture plate (TCP) wells. TCP wells containing cells cultured without beads were the negative controls. Subsequently, fresh DMEM nutrient medium (990 µl) was added to each TCP well and cells were placed in a CO2 incubator at 37°C. After 24 h incubation, cell growth and viability were assessed using the MTT assay and by measuring the absorbance of the samples at a wavelength of 490 nm at 1, 3, 5, and 7 days using a microplate reader .feration . Brieflyvia analysis of variance (ANOVA) and calculation of p-value (<0.05) as an indicator of statistical significance.All the experimental data and results were measured quantitatively and are indicated as mean ± standard deviation, with statistical analysis Bead morphology and relative size measurements (mean ± SE) were conducted through simple visualization using microscopy. The bead shape was spherical, and bead size ranged from 0.5 to 0.8 mm in diameter ; Table 2The porosity of beads indicates the diffusion rate of molecules through the cell membrane. All the beads demonstrated good porosity, and the porosity of the control sample beads was 80%. However, the porosity of the ESMP-containing beads was significantly reduced as we increased the concentration of ESMP, i.e., 80%–60%, which demonstrates the significant difference in the adsorption of the ESMP particles on the surface of the polymeric matrix .Aloe vera pulp with good water absorption capacity resulted in the removal of exudate from the wound for better healing , 1750 cm−1 (C=O stretching), 1267 cm−1 (amide III C–N group), and 1001 cm−1 (C–S); Alginate—3445 cm−1 (O–H vibration) and 1729 cm−1 (C=O stretching) for glucuronic and mannuronic acid units; Aloe vera—3438 cm−1 (O–H vibration), 2920 cm−1 (symmetric = CH2 stretching), 1721 cm−1 (C=O stretching), and 1580 cm−1 ; and ESMP˗ 3492 cm−1 , 2125 cm−1 (C–H stretching), 1635 cm−1 (amide I C=O stretching), 1416 cm−1 (N–H stretching), and 587 cm−1 (C–S stretching), respectively. In the case of control beads of alginate, orange peel, and Aloe vera, the shifting and disappearance of orange peel and Aloe vera functional peaks indicated bonding between the functional groups. However, the stretching mode formed peaks at 3487 and 1742 cm−1 for the C–O, O–H, and N–H groups, with asymmetric stretching vibration of C–H bonds. Correspondingly, the ESMP-modified bio-polymeric bead sample showed the same stretching and shifting peaks as observed in the control group. Stretching at 1635, 1416, and 587 cm−1 relates to the stretching modes of C-O, N-H, and C-S bonds. The FTIR data confirmed the complete linkage between the functional groups of bio-polymeric bead constituents and free functional groups for better cell attachment. Likewise, other scientists have fabricated biodegradable scaffold and reported peaks for all the essential proteinaceous compounds in the regions: 3200–3500 cm−1, 1600–1700 cm−1 (amide I), 1500–1600 cm−1 (amide II), and 1200–1300 cm-1 (amide III), respectively  and Gram-negative Staphylococcus aureus (S. auerus) bacterial strains suppressed growth of bacterial cells over time  enhance their therapeutic index for slowing the ageing process and are potentially applicable as regenerative medicine for bio-functional tissue. We conclude that in vivo or pre-clinical trials must be performed to prove the commercial applicability of the beads as consumer-friendly products that can be used as cosmetic or therapeutic products in the same manner as current commercially available dermatological gels, creams, face wash, scrubs, and cleansing agents.In this study, the bio-polymeric beads of alginate, orange peel,


Dear Editor,Patients with chronic lymphocytic leukemia (CLL) are at increased risk for infection due to immunosuppression that is both intrinsic to the disease and secondary to therapy. Although bacterial infections are the most common, CLL results in T-cell dysfunction that also predisposes patients to a wide variety of viral infections . TreatmeIxodes scapularis tick. Although this tick is more well-known for transmitting Borrelia burgdorferi and Anaplasma, it can also transmit the Powassan virus and is endemic to the Great Lakes region and the Northeastern United States. When severe, infection with Powassan virus can cause meningoencephalitis with a case fatality rate of 10%. Approximately half of the people who survive severe disease can experience permanent neurologic sequalae [Due to this immunosuppression, CLL patients may be vulnerable to severe infection from other opportunistic pathogens, such as endemic flaviviruses, that typically cause minimal symptoms in immunocompetent individuals. Powassan virus is an arthropod-borne flavivirus that is transmitted to humans primarily by the sequalae . We pres2O and a negative infectious workup , receiving therapy with the BTKi ibrutinib, was transferred to our institution after presenting with headache, recurrent fever, and rapidly progressive weakness following a recent hunting trip where he noticed a tick bite. He had recently completed a 10-day course of doxycycline for presumed Rocky Mountain Spotted Fever without clinical improvement. He experienced progressive ascending flaccid paralysis with bulbar weakness and involuntary head and neck movements, requiring intubation for airway protection and transfer to our institution. A lumbar puncture on admission demonstrated a lymphocytic pleocytosis with elevated opening pressure of 27 cm Hup Table . CerebroCase 2: A 75-year-old man with Parkinson’s disease and relapsed high-risk CLL del17p), on ibrutinib therapy, was transferred to our institution for headache and encephalopathy. Similar to case 1, he had recently suffered a tick bite and had been treated with an outpatient course of doxycycline without clinical improvement. Upon transfer, his exam demonstrated rigidity with saccades and vertical upgaze restriction. Carbidopa/levodopa was uptitrated given concern for progressive supranuclear palsy, but he continued to decline. MRI brain, obtained following further clinical decline with the inability to follow commands, demonstrated cerebral cerebritis and obstructive hydrocephalus requiring transfer to the Neurosurgical ICU and placement of an extraventricular drain Fig. . CSF ana7p, on ibIxodes scapularis, other Ixodes species, and Dermacentor andersoni. The incidence of neuroinvasive Powassan virus in the United States is consistent with the geographic distribution of these ticks, with concentrations in the Upper Midwest and Northeast [Since its original description in 1958, 206 cases of neuroinvasive Powassan virus infections have been reported in the U.S. through the end of 2020 –6. Casesortheast .Defects in humoral and cellular immunity due to CLL itself and its treatment result in an increased risk of infections that are a significant cause of morbidity and mortality in CLL patients . Most reTo the best of our knowledge, this represents the first report of fatal Powassan virus encephalitis among CLL patients. There is a case report of a single patient with CLL, not on treatment, who contracted Powassan encephalitis and survived, although he experienced significant morbidity, including aphasia and spastic paraplegia . Other rDue to their uniquely immunocompromised state, CLL patients are at increased risk of serious infection, and the community of providers treating CLL should be aware of this pathogen. As seen in these cases, many CLL patients develop hypogammaglobulinemia, which only further worsens infectious risk. Educating patients residing in or visiting areas where the Powassan virus is endemic about strategies to mitigate tick bites is paramount in preventing infection. This includes the application of insect repellents to the skin  plus permethrin insecticide-treated clothing.The lack of a widely available molecular test may hamper the detection of the Powassan virus early in its course. Current testing options include IgM ELISA serologies (confirmed with plaque reduction assays), reverse-transcriptase PCR (RT-PCR) testing of serum, CSF, or tissue samples, immunohistochemistry testing of tissue, and metagenomic next-generation sequencing. Furthermore, for patients with CLL or other causes of immunocompromise in which Powassan virus encephalitis is in the differential, it is important to understand that they may be unable to mount an antibody response, potentially resulting in false negative Powassan virus IgM serologies. Such was the case with one of our patients and has been reported in other clinic contexts . MetagenIn summary, we report two patients with high-risk CLL who were on ibrutinib therapy and developed worsening neurological symptoms following a tick bite. The constellation of clinical presentation, MRI findings, and CSF lymphocytic pleocytosis in the absence of malignant cells led to a diagnosis of viral encephalitis. Further CSF studies confirmed a diagnosis of Powassan virus encephalitis—which despite maximal supportive care, led to our patients’ deaths.https://www.cdc.gov/ticks/avoid/on_people.html) is of utmost importance.These cases illustrate the importance of keeping Powassan virus encephalitis in the differential diagnosis of CLL patients who reside in the Midwest, and other endemic areas of this infection, and who present with signs and symptoms of encephalitis. In the absence of definitive therapy and given poor outcomes following Powassan virus infection, adherence to CDC guidelines to prevent tick bites (Supplemental Figure 1.Supplemental Figure Legend


A new H7N9 virus of avian origin crossed the species barrier to infect humans, causing high mortality and emerged as a potential pandemic threat. The mechanisms underlying the virulence and pathogenicity of H7N9 virus remains elusive. H7N9 virus originated from a genetic assortment that involved the avian H9N2 virus, which was the donor of the six internal genes. Unlike the H7N9 virus, the H9N2 virus caused only mild phenotype in infected mice. In this study, we used the mouse infection model to dissect the difference in the host response between the H7N9 and H9N2 viruses. Through analyzing transcriptomics of infected lungs, we surprisingly found that the H9N2 infection elicited an earlier induction of innate immunity than H7N9 infection. This finding was further corroborated by an immunohistochemical study demonstrating earlier recruitment of macrophage to the H9N2-infected lung than the H7N9-infected lung, which could occur as early as 6 hours post infection. In contrast, H7N9 infection was characterized by a late, strong lung CD8+ T cell response that is more robust than H9N2 infection. The different pattern of immune response may underlie more severe lung pathology caused by H7N9 infection compared to H9N2 infection. Finally, we could show that co-infection of the H9N2 virus protected mice from the challenge of both H7N9 and PR8 viruses, thereby strengthening the importance of the induction of an early innate immunity in the host’s defense against influenza infection. Collectively, our study unraveled a previously unidentified difference in host response between H7N9 and H9N2 infection and shed new insight on how virus-host interaction shapes the  Since the first virus strain was identified in 1930s, influenza A viruses (IAVs) continue to pose a threat to humans. The IAV challenge, at least in part, stems from the fact that multiple hosts exist, including a variety of avian species, pigs, and humans, and new viruses can emerge from reassortment when two or more viruses infect the same cell. Normally the species barriers would prevent IAV’s spillover from animal to human. However, such a barrier could be broken, resulting in human exposure to new viruses to which they do not have immunity, and consequently can lead to high mortality and even a pandemic if the virus evolves into acquiring human-to-human transmission ability. Most recently, a case was found in a novel avian IAV of H7N9 subtype (abbreviated as H7N9 throughout this paper), which was first reported to cause severe human respiratory infection with high fatality rate in Shanghai and Anhui Province, China, in March 2013 . PhylogeThe immune response of the host, comprised of two arms, innate immunity and adaptive immunity, is crucial in determining disease pathogenesis caused by viral infection and is the basis for the development of control strategies . While aThere have been several published studies on the characterization of H7N9 virus in mice. These studies revealed that H7N9 virus caused more severe phenotype than H9N2 virus and seasonal H3N2 influenza virus, which correlates to increased infectivity and elevated induction of proinflammatory cytokines . Of inteOur group has adopted a comparative approach to understanding the mechanisms underlying mammalian adaptation and pathogenesis of the H7N9 virus using H9N2 as a counterpart. Through performing bulk transcriptomic profiling of lung tissues from virus-infected mice, we recently identified IFN-κ as one of the earliest Type I IFNs induced by the H9N2 infection whereas this early induction was not featured in response to H7N9 infection . This fiA/Shanghai/4664T/2013(SH/4664) H7N9 virus (Genbank accession number: KC853225.1-KC853232.1) was conserved at a Biosafety Level 3 (BSL3) lab at Shanghai Public Clinical Center. A/Chicken/Shanghai/F/98 (CK/F98) H9N2 virus  was conserved at Shanghai Veterinary Research Institute, Chinese Academic of Agricultural Sciences.via intraperitoneal injection of ketamine (100mg/kg), and then intranasally inoculated with a dose of 1× 106 EID50 (50% egg infectious dose) virus, or PBS in a volume of 50μl. The infected mice, where indicated, were divided into subgroups for different analyses; for the assessment of morbidity and mortality, the mice were monitored for survival and body weight changes over a 14-day observation period. Mice with 30% or more body weight loss were recorded dead and humanely euthanized. For the co-infection experiment, mice were intranasally inoculated with 1x104 TCID50 of H7N9/PR8, or 1.7x107 EID50 of H9N2, or both viruses in a 50µl volume. All the animal-related experiments were carried out in a BSL3 facility at Shanghai Public Clinical Center. This study was performed in accordance with Home Office guidelines and were approved by the Shanghai Public Health Center Local Ethical Committee.Eight- to ten-week-old female C57BL/6 mice  were maintained under specific pathogen-free conditions at the animal facilities of Shanghai Public Health Clinical Center. For the animal study comparing virulence of H7N9 versus H9N2, mice were randomly grouped, anesthetized Mice were sacrificed at 6 hours, day 1, day 2, day 3, and day 7 after the virus challenge for collection of lung tissues. Lung tissues were fixed with 4% paraformaldehyde and then embedded in paraffin. Five-micrometer sections were cut and stained with H&E. The pathology scores were calculated following a methodology we previously published . For immEach lung from the H7N9/H9N2 infected mice was mechanically and ultrasonically homogenized at 4°C in 1.2ml RNAzol . Total RNA was extracted from homogenized tissue according to the protocol supplied by the manufacturer. The resulting RNA preparations were analyzed on a 2100 Bioanalyzer  to ensure quality and integrity. For microarray hybridization, 500ng of RNA were applied to Cy3-labelling reaction using the one-color Quick Amp Labelling protocol and the labeled cDNAs were hybridized to Agilent’s mouse 8 x 60 k microarrays followed by detection using the Agilent Scanner G2505C (Agilent Technologies). The microarray data were analyzed by RBR embedded in Excel and cluster analyses were performed using MEGA5. The microarray data were deposited in Gene Expression Omnibus (GEO) under accession number GSE142709.Total RNA was isolated from homogenized lung tissue using an RNA isolation kit . Reverse transcription was performed using oligo (dT) primer and the Reverse Transcription System , and SYBR green-based real-time PCR was performed on the resulting cDNAs using Mastercycler reprealplex real-time PCR system . The data were analyzed by the REALPLEX2.2 software. The gene-specific primers used in this study were: mCXCL10: F-5’-GGTCTGAGTCCTCGCTCAAG-3’, R-5’-GTCGCACCTCCACATAGCTT-3’; mIL1β: F-5’-GAAATGCCACCTTTTGACAGTG-3’, R-5’-CTGGATGCTCTCATCAGGACA-3’; mIL6: F-5’-CTGCAAGAGACTTCCATCCAG-3’, R-5’-AGTGGTATAGACAGGTCTGTTGG-3’; mTNFα: F-5’-CATCTTCTCAAAATTCGAGTGACAA-3’, R-5’-TGGGAGTAGACAAGGTACAACCC-3’; mGAPDH: F-5’-TGGCCTTCCGTGTTCC TAC-3’, R-5’-GAGTTGCTGTTGAAGTCGCA-3’.in situ hybridization, we chose viral nucleoprotein (NP) RNA as target RNA for detection because of its high expression in infected cells and high conservation among different virus strains. An NP-specific probe, namely Probe-NP, is customized to target 236-1367 region of the NP gene of the influenza virus A/Shanghai/1/2013 (H7N9) (GenBank: KF609528.1). Sequence comparison confirmed that the targeted NP gene sequence is highly conserved in the H9N2 virus. The preparation of slides and RNA in situ hybridization using RNAscope reagents (Advanced Cell Diagnostics) were performed as described previously . Data were expressed as mean ± SEM. One-way analysis of variance (ANOVA) test was applied when comparing more than two groups. A 6 EID50 of either the H7N9 or H9N2 virus, and monitored over a period of 14 days for survival and weight loss. Part of the group was subjected to lung viral load determination at 4-days post infection (dpi) by titration in embryonated eggs. The H7N9 challenged group showed a sustained weight loss starting from 1 dpi, whereas the H9N2 challenged group experienced a moderate weight decrease during the first 7 days and regained weight afterwards (4 EID50/g versus 1.78 × 106 EID50/g)  or a dose of 1 × 10in situ hybridization technology (RNAscope) to assess the dynamic distribution of virus-infected cells, indicated by the presence of viral NP RNA, in the lung. Cells positive for NP RNA (NP-positive cells) could be detected in both bronchi and alveoli of the lung section from H7N9 infected mice 6 hours post infection (0.25 dpi) and saw a marked increase in the number over 1-3 dpi, followed by a sharp decline observed at 7 dpi. The number of NP-positive lung cells were significantly lower in samples from the H9N2 group, compared to those of H7N9 group at all time points until becoming undetectable at 7dpi  level, and then underwent a sharp decline by 7 dpi  and CXCL12. The analyses revealed that, compared to the H7N9 infection, the H9N2 infection induced an earlier induction of both CCR2 and CXCR4 transcripts, similar to that of CCL2 , as wellWe also probed the dynamics of the T cell immune response reacting to H9N2 and H7N9 infections. The results showed a more robust CD8+ T cell response in the lung induced by the H7N9 infection than H9N2 infection. Importantly, for the H7N9 infection, such T cell response showed a pattern of gradual increase with a peak attained on day 2 post infection. Our previous studies of hospitalized H7N9 patients revealed the virus resolution and survival depends on a diversity of response mechanisms, wherein an early prominent H7N9-specific CD8(+) T-cell responses is translated into a shorter discharge time and increased survival. This rapid CD8(+) T cell response is potentially derived from cross-reactive memory T cells deposited by prior influenza exposure . UtiliziFinally, we performed a co-infection experiment to examine our hypothesis that the elicitation of an early innate immunity is critical for protection against influenza virus infection. We could show that co-inoculation of H9N2 did not enhance the lethality and pathogenicity of the host, but rather reduced the pathogenicity of H7N9 infection. An even better protective effect was observed with the PR8 virus. The lesser efficacy of H9N2-mediated protection against H7N9 versus PR8 might be due to a relatively higher replicative capacity of the H7N9 virus. Given that H7N9 and PR8 viruses express very different HA and NA surface proteins, the ability of H9N2 co-infection to protect mice against the challenge of a heterologous influenza virus is consistent with a mechanism primarily mediated by innate immunity, as we proposed. Taken together, we think that the lesser pathogenicity of H9N2 in the mouse model has dual mechanism: first, it is intrinsically attenuated when infecting mammalian cells and our previous study has pinpointed such defect to the lack of mammalian adaptation of its internal genes, most likely its polymerases genes ; secondlIn summary, we presented here a comparative analysis of immune responses to the H7N9 virus versus the H9N2 virus. This analysis, together with a following demonstration of the protection against H7N9 and PR8 challenge by H9N2 co-infection, brings a new perspective to the interaction between host immunity and influenza evasion. That is, the induction of early innate immune response, as seen with in H9N2 infection but absent in H7N9 infection, is the first but also potentially a primary step toward an effective viral inhibition. Without this step, adaptive immunity could be responsible for viral clearance; however, a sustained CD8+ T cell response may leave the host at higher risk of lung immunopathology. There are some important questions warranting future investigation. For example, what is the mechanism mediating H7N9’s escape from trigging an early innate immune response? Is there any specific viral factor involved? An equally important question is to identify the pathogen-associated molecular pattern (PAMP) pathway(s) responsible for the detection of H9N2 infection. It should be noted that the importance of such a pathway would not be unraveled in the case of H7N9, where a viral evasion mechanism might evolve. Furthermore, is avoiding early innate immunity a general mechanism utilized by other influenza viruses that cause severe diseases? Conversely, a comparative measurement of pulmonary specific compliance and the composition of bronchoalveolar fluid is a worthy effort that has the potential to provide new clues into the H7N9-caused lung pathology and the underlying causes. These future investigations would certainly help us further understand the complex host-influenza interactions, thereby providing a new theoretical basis for development of new therapeutics for treatment of influenza-related human diseases.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by Shanghai Public Health Center Local Ethical Committee.in vivo experiments. All authors contributed to the article and approved the submitted version.XZ and JX conceived and designed this project and supervised the experiments. CZ and YY performed the IHC and IF experiments. WF performed extraction of total RNA and microarray analysis. MZ performed virus titer determination. YH, LZ, SY and LJ performed the This work was supported by the National Natural Science Foundation of China  , the Intramural Funding from Shanghai Public Health Clinical Center , Shanghai Science and Technology Program (20Y11900500), and Sichuan Science and Technology Program (2021YFS0404 and 2021ZYD0082).We thank Professor Zejun Li of Shanghai Veterinary Research Institute, Chinese Academic of Agricultural Sciences for providing virus.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Totiviridae, Endornaviridae, and Partitiviridae, greatly expanding the reported host range for these families. Two new species belonging to the single-stranded RNA viral clade Marnaviridae, commonly associated with microalgal hosts, were also identified. This study highlights that a substantial diversity of RNA viruses likely exists undetected within the unicellular microalgae. It also highlights the necessity for RNA viral characterization and for investigation of the effects of viral infections on microalgal physiology, biology, and growth, considering their environmental and industrial roles.Unicellular microalgae are of immense ecological importance with growing commercial potential in industries such as renewable energy, food, and pharmacology. Viral infections can have a profound impact on the growth and evolution of their hosts. However, very little is known of the diversity within, and the effect of, unicellular microalgal RNA viruses. In addition, identifying RNA viruses in these organisms that could have originated more than a billion years ago constitutes a robust data set to dissect molecular events and address fundamental questions in virus evolution. We assessed the diversity of RNA viruses in eight microalgal cultures, including representatives from the diatom, eustigmatophyte, dinoflagellate, red algae, and euglenid groups. Using metatranscriptomic sequencing combined with bioinformatic approaches optimized to detect highly divergent RNA viruses, we identified 10 RNA virus sequences, with nine constituting new viral species. Most of the newly identified RNA viruses belonged to the double-stranded IMPORTANCE Our knowledge of the diversity of RNA viruses infecting microbial algae—the microalgae—is minimal. However, describing the RNA viruses infecting these organisms is of primary importance at both the ecological and economic scales because of the fundamental roles these organisms play in aquatic environments and their growing value across a range of industrial fields. Using metatranscriptomic sequencing, we aimed to reveal the RNA viruses present in cultures of eight microalgae species belonging to the diatom, dinoflagellate, eustigmatophyte, rhodophyte, and euglena major clades of algae. Accordingly, we identified 10 new divergent RNA virus species belonging to RNA virus families as diverse as the double-stranded Totiviridae, Endornaviridae, and Partitiviridae and the single-stranded Marnaviridae. By expanding the known diversity of RNA viruses infecting unicellular eukaryotes, this study contributes to a better understanding of the early evolution of the virosphere and will inform the use of microalgae in industrial applications. Viruses are often considered the most ancient “life forms” . As studies of the viromes of increasingly diverse taxa proceed, the more their remarkable ubiquity, diversity, and abundance becomes apparent . RNA vir4–Knowledge of the RNA virosphere in overlooked eukaryotic lineages that evolved billions of years ago—such as the microalgae—could greatly enhance our understanding of the earliest events in RNA virus evolution. With barely 100 species of RNA viruses reported since the first isolation of a microalgae-infecting RNA virus in 2003 , our curMarnaviridae are the best described family of microalgae-infecting viruses , Durnavirales (Partitiviridae-like), and Martellivirales (Endornaviridae-like) , Nodamuvirales (Nodaviridae), Wolframvirales (Narnaviridae), and Cryppavirales (Mitoviridae) phyla , the metagenomic era has multiplied the number of RNA viruses described by many thousands –13. This–14– viruses , 22, 23,ae-like) , 24–26, e) phyla , 28. To e) phyla , 28–30 aRhizaria , 20, ChlRhizaria , 31, RhoRhizaria , 26, andNannochloropsis oceanica and Nannochloropsis oculata) and diatoms , alveolates including dinoflagellates , red algae (Rhodella maculata), and euglenid (Euglena gracilis). By using a “culture-based” metatranscriptomic approach, we combined the power of unbiased detection of ultralarge-scale RNA sequencing with the use of monoorganism culture to assist in associating the viruses identified to their specific algae hosts. Given the high levels of sequence diversity observed in many RNA viruses, we paid particular attention to identifying divergent virus-like sequences.To increase our understanding of the RNA virosphere in microalgae, we assessed the diversity of RNA viruses in eight microalgal species, covering the major groups of stramenopiles, including eustigmatophytes  sequence did not return any PCR signal -PCR. This resulted in the detection of 8 of the 11 virus/samples tested . Triopascum RNAs , likely cum RNAs . MegareuR signal . Hence, cultures  may explDuplornaviricota viruses, two falling into the Kitrinoviricota viruses, and six sharing homologies at amino acid level with Pisuviricota viruses  sequences at the phylum level using the recently developed RdRp-scan resource . These l viruses .Ghabrivirales, Endornaviridae, Durnavirales, and Marnaviridae lineages and are described below.We then conducted additional phylogenetic analyses focusing on the viral subclades that contained the 10 newly identified sequences. These comprised the Totiviridae-like viruses  within the Totiviridae  samples  of the diatom Phaeodactylum tricornutum, with only eight single-nucleotide polymorphisms (SNP) reported at the genome level.A second Toti-like virus,  samples . They alnd fungi –35. To cTaphios ghabri-like virus 1 sequence, at 4.8 kb, is in the range of other Totiviridae and, along with the read coverage profile, suggests that the full-length genome has been obtained , and commercial cultivation yields.The observation of croalgae  and uniccroalgae . Notablyir hosts –40. The Endornaviridae family of dsRNA viruses. Specifically, Althepos endorna-like virus 1 and Almopos endorna-like virus 1 (both retrieved from G. carpenteri–Dinophyceae) group with members of the Alphaendornavirus genus, a genus within the Endornaviridae previously associated with land plants, fungi, and oomycetes EndornaviEndornaviridae dsRNA genomes are 9.7 to 17.6 kb in length and encode a single polyprotein with a RdRp domain located in the C terminus , except for its genome size of ~21 kb, which is the longest genome reported to date for this group. In addition to the viral RdRp domain located in the C-terminal region of the Almopos endorna-like virus 1 protein, other protein domains and signatures could be identified that were related to the (+)RNA virus helicase core (IPR027351), the YbiA-like superfamilies (IPR037238), and the UDP-glycosyltransferase/glycogen phosphorylase superfamily (SSF53756) , similar to previous studies . Whether Althepos endorna-like virus 1 and Almopos endorna-like virus 1 affect the fitness of their G. carpenteri host remains to be investigated but may have important implications for the management of this potentially harmful species  and G. carpenteri (Dinophyceae) cultures, respectively, form a clade with the Ourea durna-like virus previously associated with the dinoflagellate Dinophysis acuminata Orion durd plants  and reced plants .Orion durna-like virus 1 and D. durna-like virus 1 genomes  with a single ORF containing the RdRp domain . Partitiviridae are bisegmented viruses. Considering the placement of Orion durna-like virus 1 and D. durna-like virus 1 within the Partitiviridae phylogeny, it is very likely that they comprise a second segment, potentially encoding a coat protein not retrieved in this study due to our RdRp-based retrieval methodology. A complementary comparison of those sequences in the SRA database identified a sequence with 100% sequence identity at the amino acid level to D. durna-like virus 1 from a Gambierdiscus polynesiensis (dinoflagellate) sample (SRR3358210) (Table S2).Partitiviridae genus that is specific to dinoflagellates, comprising Orion durna-like virus 1, D. durna-like virus 1, and the previously identified Ourea durna-like virus. This observation expands the host range reported for this family, already comprising plants, fungi, oomycetes, apicomplexan parasites, and green algae  sample and exhibited RdRp sequence similarity with the Marnaviridae, a picorna-like family of ss+ RNA viruses that infect unicellular eukaryotes Minyas maaviridae .Minyas marna-like virus 1 and Megareus marna-like virus 1 and their average read coverages  and mitochondrial (GCA_001638955) revealed strong identities (Table S1). A very close sequence (seven SNPs at the genome level and six nonsynonymous substitutions at the RdRp level) could also be retrieved from the SRA database sample (SRR2294740), corresponding to the mitochondrial genome of E. gracilis. Such a mitochondrial sublocation is also suggested by the ORF found that can be expressed using the Chlorophycean mitochondrial genetic code . Hence, this Pisuviri-like signal appears to be part of the host genome, likely corresponding to an endogenous viral element. The presence of such EVEs will help identify divergent viruses infecting euglenoid lineages, which are expected to be highly divergent, considering the basal placement of euglenoids within eukaryotic organism diversity . No functions could be associated with the additional ORFs, and further studies are required to characterize this newly identified protist-infecting virus.The R. maculata was retrieved in very high quantity and identical to the D. mito-like virus .While many unassigned entries in most of the samples analyzed here can limit the formal assignment of viruses to hosts, obtaining a clearer picture of the eukaryotic host sequences present in the sample and their relative abundance can help to discriminate between eukaryotic hosts. Indeed, our cultures were washed several times before RNA extraction. It is therefore likely that the viral sequences identified result from intracellular viral forms rather than extracellular virions circulating in the culture media: hence, we assume that viruses detected in this study are associated with cellular organisms that are also present in the sample. We therefore examined the deep taxonomy of BLASTx eukaryotic-like contigs, as well as the total contig abundance reported for major eukaryotic lineages , which hrpenteri  could coP. pisuviri-like virus 1 identified from R. maculata, the majority of detectable contigs belong to the corresponding Rhodophyta host taxa, suggesting that this virus is likely associated with a Rhodophyte host rather than fungi or other contaminant organisms. The very large proportion of contigs associated with land plants (Streptophyta) in the N. oculata library might correspond to contamination. However, the unambiguous placement of the corresponding Minyas marna-like virus 1 virus within the well established microalgae-infecting Marnaviridae provides a strong argument that this virus is associated with diatoms.In the case of the Of note, all of the viruses reported here were identified in healthy algae cultures and therefore assumed to be detrimental to their algal hosts. It will be of considerable interest to characterize the phenotypic effects of such seemingly commensal infections, whether persistent, neutral, or even beneficial, and to understand their underlying molecular basis.Totiviridae and Partitiviridae, as well as the enrichment of the positive single-stranded picorna-like family Marnaviridae. This study also extended the host range of the dsRNA endornaviruses to microalgae, raising questions about how this viral family is able to infect the plant, fungi, and TSAR eukaryotic supergroups. Considering the harmful or commercial value of their hosts, this description of new microalgal viruses paves the way for further studies of the effects of viral infections on host biology and their associated ecological and industrial consequences. Finally, this study highlights the need to reveal the hidden diversity among RNA viruses infecting microalgae and among microbial eukaryotes in general, particularly considering their fundamental and applied importance.Through metatranscriptomic sequencing of total RNA from microalgae cultures we identified 10 new RNA viruses associated with diatom, eustigmatophyte, dinoflagellate, and rhodophyte microalgae. These newly discovered viruses contribute to the establishment of new microalgae-infecting viral clades within the −2 s−1. Culture media and temperature conditions were specific to each species and were as follows: N. oceanica 24°C, f/2 medium; N. oculata 24°C, f/2 medium; T. weissflogii 20°C, f/2 medium; R. maculata 24°C, L1 medium (minus Si);E. gracilis 20°C, euglena medium; Prorocentrum cf. balticum (UTSPH2D4) (P. lima 25°C modified K medium (G. carpenteri (UTSHI2C4) 25°C modified K medium  ; 20°C K K medium ; and G. K medium , 59. To T. weissflogii) and Euglenozoa (E. gracilis) cultures were extracted using the RNeasy Plus Universal kit (Qiagen), according to the manufacturer’s instructions. Qiazol lysis buffer was then added to frozen pellets, and homogenization was performed by pipetting. Genomic DNA was removed, and RNA was extracted using 1-bromo-3-chloropropane. Supernatants were then transferred to Qiagen columns. After washing the columns, pure RNAs were collected into sterile water strictly following kit instructions.Total RNA from the diatom , the Rhodophyta R. maculata, and the eustigmatophyte (N. oceanica and N. oculata) cultures was extracted using Allprep DNA/RNA kit (Qiagen), following the manufacturer’s instructions. Briefly, frozen cell pellets were supplanted with lysis RLT buffer and cells disrupted using bead beating with 0.5-mm glass beads. An additional step of sample homogenization using QIAshredder (Qiagen) was added during the RNA extraction of R. maculata sample and N. oceanica to reduce the viscosity of eluates. Cell debris was removed using a centrifugation step at high speed, and the supernatants were transferred to Qiagen columns. Total RNA fractions were then purified after several washing steps and eluted according to the kit instructions.Total RNA from dinoflagellates . The corresponding libraries were sequenced on the NovaSeq platform (Illumina)  by the AGRF.RNA quality was checked using a TapeStation and individually converted by the Australian Genome Research Facility  into non-rRNA RNA-seq libraries using TruSeq Stranded Total RNA with Ribo-Zero Plant (Illumina). Due to the very low RNA yields obtained for Total reads were filtered using Trimmomatic (version 0.36)  to removTo help determine the taxa composition of each library, all contig sequences were compared to the nonredundant protein database nr from NCBI using Diamond BLASTx (version 2.0.9)  and to t(i) Sequence-based similarity detection. RdRp sequences corresponding to RNA viruses  were first identified by comparing contigs to the nr database using Diamond BLASTX  ( < 1e−5) . To maxi < 1e−5) , were us(ii) HMM-based homology detection of ORFans. All orphan contig sequences  were compared to the RdRp HMM profiles of the RdRp-scan resource (resource  and usinresource .(iii) Genome extension, genome coverage, and virus annotation. To ensure all the RNA virus-like sequences could be identified and in their longest form, additional attempts to assemble contigs were performed using the rnaSPADES (version 3.13.0) ( 3.13.0)  and Mega 3.13.0) . This di 3.13.0) . A virus 3.13.0)  and RdRp 3.13.0) . Genome  3.13.0)  and Samt 3.13.0) . The res 3.13.0) . The ove(iv) SRA mining. To help retrieve complete genome sequences, assess intraspecies variability, and associate viruses with particular algae hosts, we performed an additional step of Sequence Read Archive (SRA) mining for each of the 10 new viruses identified in this study. For each algae library, we screened the SRA using nucleotide Magic-BLAST (version 1.3.0) (n 1.3.0) . When thn 1.3.0)  and SAMtn 1.3.0) .(v) Phylogenetic analysis. RNA virus phyla-level comparisons were performed using Clustal Omega (version 1.2.4) (n 1.2.4)  to direcn 1.2.4) . Initialn 1.2.4) . Subalign 1.2.4)  and manun 1.2.4) . Maximumn 1.2.4)  with then 1.2.4)  and usinTo experimentally confirm viral contigs assembled from RNA-seq data, cDNAs from each of the total RNAs were first obtained using the SuperScript IV reverse transcriptase (Invitrogen). PCRs were then performed on each cDNA sample using corresponding host and virus primers (detailed in Table S3) using the Platinum SuperFi II DNA polymerase (Invitrogen) and following the manufacturer’s instructions.PRJNA867582, with accessions SAMN30215649 to SAMN30215656. The viruses newly identified here are available at GenBank/NCBI under the accessions OP191686 to OP191695.Corresponding RNA-seq read files are available on the SRA under BioProject


Giraffa spp.) populations, has emerged as a potential conservation threat. Preliminary findings suggested that individuals with GSD lesions move with greater difficulty which may in turn reduce their foraging efficiency or make them more vulnerable to predation. A current known threat to some giraffe populations is their mortality associated with entrapment in wire snares, and the morbidity and potential locomotor deficiencies associated with wounds acquired from snares. The goal of our study was to quantify the locomotor kinematics of free-ranging Nubian giraffe  in Murchison Falls National Park (MFNP), Uganda, and compare spatiotemporal limb and neck angle kinematics of healthy giraffe to those of giraffe with GSD lesions, snare wounds, and both GSD lesions and snare wounds. The presence of GSD lesions did not significantly affect spatiotemporal limb kinematic parameters. This finding is potentially because lesions were located primarily on the necks of Nubian giraffe in MFNP. The kinematic parameters of individuals with snare wounds differed from those of healthy individuals, resulting in significantly shorter stride lengths, reduced speed, lower limb phase values, and increased gait asymmetry. Neck angle kinematic parameters did not differ among giraffe categories, which suggests that GSD neck lesions do not impair normal neck movements and range of motion during walking. Overall, MFNP giraffe locomotor patterns are largely conservative between healthy individuals and those with GSD, while individuals with snare wounds showed more discernible kinematic adjustments consistent with unilateral limb injuries. Additional studies are recommended to assess spatiotemporal limb kinematics of giraffe at sites where lesions are found predominantly on the limbs to better assess the potential significance of GSD on their locomotion.Giraffe skin disease (GSD), a condition that results in superficial lesions in certain giraffe ( Giraffa spp.) have declined in both distribution and abundance over the last century due to habitat loss and fragmentation, civil unrest, poaching , and ecological change4. Giraffe populations experienced an overall ~ 30% decline in the last three and a half decades, and today there are an estimated 117,000 giraffe in the wild3. In 2016, the International Union for Conservation of Nature (IUCN) up-listed giraffe as a single species  from “Least Concern” to “Vulnerable” on the Red List, emphasizing the population declines and severity of threats facing them. The taxonomic classification of giraffe is a topic of debate12; however, here we utilize the classification that recognizes four taxonomically distinct species: Masai giraffe (G. tippelskirchi), reticulated giraffe (G. reticulata), northern giraffe , and southern giraffe (G. giraffa)12. The conservation status of three species  are of great concern, with their numbers all declining by > 50% and absent from much of their estimated historic geographical ranges13. Giraffe play a key role in shaping the ecology of savannah and woodland ecosystems, and their loss across the continent could have far-reaching long-term ecological consequences15.Once widely distributed across the continent of Africa, giraffe  in Uganda16. Since then, GSD has been detected in at least 13 parks and reserves across seven countries in Africa, including South Africa, Botswana, Namibia, Zimbabwe, Kenya, Tanzania, and Uganda17. Prevalence of GSD has been reported to be as high as 86% in Masai giraffe inhabiting Ruaha National Park (NP), Tanzania20, and at a given time, as many as 51% of giraffe in Murchison Falls NP (MFNP) in Uganda may be afflicted with visible signs of GSD . Furthermore, skin conditions resembling GSD have been reported in 11 zoos in six countries, including Belgium, England, France, Italy, the Netherlands, and the United States17. We now know that GSD manifests as different skin conditions; however, all GSDs generally manifest as large, crusted, scab-like wounds, sometimes with accompanying cracks in the skin, appearing mainly on the limbs, chest, and neck, though location of lesions differs by geographical location23  emerged as a new potential threat to giraffe conservation. GSD was first described in a population of Nubian giraffe , a disease that affects locomotor mechanics and efficiency could have serious consequences for an individual’s ability to seek resources25 and evade predators27.Several etiological agents have been suggested in the pathogenesis of different GSDs, including bacterial, fungal, and protozoal organisms, although current research suggests a nematode origin, with possible accompanying fungal infection28. The relatively indiscriminate nature of snares results in a considerable amount of unintended bycatch30, which includes giraffe31. In addition to the mortality associated with poaching, injuries associated with escaping from snares have been shown to affect locomotion and behavior in other taxa32 and have been linked to increased parasite load33. In giraffe, modified locomotion and behavior could potentially increase predation risk and decrease foraging efficiency. In this way, evaluating the effects of snare wounds on giraffe locomotion could lead to a deeper mechanistic understanding of snare-related mortality and morbidity.Another threat to some giraffe populations is morbidity associated with snare-related injuries and potential mortality caused by entrapment in wire snares Fig. B. The in39. These important studies provide the groundwork for our investigation. Recently, Basu et al.39 quantified spatiotemporal limb kinematics, kinetics , and neck angle kinematics of walking gaits in three adult zoo-housed reticulated giraffe. They found that giraffe walking gaits can be classified as lateral sequence, lateral couplets . This gait type helps to avoid limb interference in which limbs contact one another during the stride but differs from a true “pace” gait where the touchdowns and liftoffs of ipsilateral limbs is simultaneous or approximately simultaneous40. The movement of the neck is functionally linked to the giraffe walking gait, with horizontal acceleration of the neck out of phase with the horizontal acceleration of the trunk39.A handful of studies have examined the locomotion and gait of giraffes18, we predicted that the spatiotemporal limb kinematics and neck angle kinematics of individuals with GSD would differ from those of healthy individuals. We also examined the mechanisms through which snare wounds impacted giraffe locomotion and predicted that, compared to healthy giraffe, individuals with snare wounds would exhibit slower walking speeds and kinematic adjustments consistent with reduced loading on the injured limb41. To our knowledge, our study is the first to quantify walking gait kinematics in free-ranging giraffe and the first to test whether and how GSD and snare wounds affect giraffe locomotion. Given the prevalence of GSD and intense wire snaring pressure on giraffe in MFNP, gaining a better understanding of how GSD and snare wounds impact locomotor capabilities of afflicted giraffe will provide important context for assessing broader health and fitness.The goal of our study was to quantify the spatiotemporal limb kinematics and neck angle kinematics in free-ranging Nubian giraffe in MFNP. In doing so, we compared kinematic parameters of healthy free-ranging giraffe  to those of free-ranging giraffe with GSD, snare wounds, and both GSD and snare wounds. Based on field observations that some giraffe with GSD were found to walk with greater difficultyWe first developed and piloted our methods in a zoo setting at Cleveland Metroparks Zoo, Ohio, USA, before applying them to videos of free-ranging Nubian giraffe from MFNP. We analyzed the gait of four Masai giraffe at Cleveland Metroparks Zoo including: one adult male (aged eight) and three adult females . The subjects were known to be in good health during the time of observation. We filmed giraffe at 30 frames per second walking over flat ground using a Casio EX-FC150 camera. Thirteen video clips containing 31 strides were suitable for spatiotemporal limb kinematic analyses , and seven strides were suitable for neck angle kinematic analyses .42. We identified each individual using their unique pelage patterns43. Videos were recorded opportunistically during ongoing conservation science research, which included assessing the etiology of GSD locally23. All videos were recorded at ~ 75–100 m from the focal animal. Individuals were recorded on flat ground to control for gait adjustments driven by changes in terrain and inclination/declination. Each giraffe was assigned to one of four condition categories: (1) healthy , (2) GSD (presence of GSD lesions), (3) snare (presence of snare wound), or (4) GSD and snare (presence of both GSD lesion(s) and snare wound). We acknowledge that giraffe assigned to our healthy category may have underlying health issues other than GSD, and some historical snare wounds are not easily observable; however, we assumed these potential factors did not significantly impact the kinematic variables examined in our study. Additionally, we recognize that researchers have categorized the severity of GSD22; however, due to limitations with our sample size, we opted to report presence vs. absence of GSD as other studies have done44.We collected data on 52 adult male giraffe in MFNP  from July 2015–August 2016. In association with a long-term study designed to evaluate the demography and spatial ecology of male giraffe in MFNP, a series of fixed route individual based photographic surveys were conducted over the entire extent of the park45. Because angular measurements are susceptible to parallax issues, only strides in which giraffe were moving approximately perpendicular to the camera were used to quantify neck angle measurements (n = 71 strides) . The videos included in the spatiotemporal gait analyses ranged from eight to 49 s and contained between one and eight strides per individual (n = 115 strides). We attempted to film perpendicular to the line of travel when possible; however, parallax is not an issue for timing and digitizing limb touchdown and liftoff events. Furthermore, our spatial points  were all digitized in the same video frame for a given video clip, eliminating potential distortion due to parallax issues. That is, all in-plane linear distance metrics would suffer a similar degree of distortion, allowing ratios of these distances to be unbiased45 . We recorded neck angle measurements for individual videoframes using the angle tool in ImageJ46.We used GaitKeeper, an open-source MATLAB package, to digitize limb liftoff and touchdown events, stride length, and shoulder height41.We quantified giraffe shoulder height (in pixels) by digitizing a point at roughly the height of the glenohumeral joint and another point at ground level directly below the shoulder. We quantified stride length (in pixels) by digitizing the initial touchdown of a reference limb  and the subsequent reference limb touchdown. Relative stride length was calculated by dividing stride length in pixels by shoulder height in pixels. Relative  stride length was reported to account for differences in body size among giraffe. We predicted that giraffe with GSD and/or snare wounds would have shorter relative stride lengths than healthy individuals41.Stride duration was recorded as the amount of time (in seconds) between the initial and subsequent touchdown of a reference limb. We used stride durations for each limb to generate mean stride duration. We predicted that giraffe with GSD and/or snare wounds would have greater mean stride durations than healthy individuals41.We calculated relative speed by dividing relative stride length by mean stride duration, resulting in values with units of % of shoulder height per second. Relative speed was reported to control for potential speed differences due to differences in body size among giraffe. We predicted that giraffe with GSD and/or snare wounds would move at slower relative speeds compared to healthy individuals47 and vary among species due to differences in anatomy and habitats and can vary within a species or individuals depending on the speed of movement and the substrate or terrain in which the animal is moving. These limb phase values are often used to classify symmetrical gaits such that limb phase values between 0 and 25% are designated as LSLC gaits, values between 25 and 50% are lateral sequence, diagonal couplet (LSDC) gaits, values between 50 and 75% are diagonal sequence, diagonal couplet (DSDC) gaits, and values between 75 and 100% are diagonal sequence, lateral couplets (DSLC) gaits. While researchers utilize different thresholds for named gait types, limb phase values equal to or approximately equal to 0%, 25%, 50%, and 75% are classified as pace, lateral sequence singlefoot, trot, and diagonal sequence singlefoot gaits, respectively48. Our preliminary data revealed that Masai giraffe housed at Cleveland Metroparks Zoo exclusively used LSLC walking gaits—a pattern consistent with another recent study of zoo-housed reticulated giraffe39. We predicted that free-ranging Nubian giraffe would also use LSLC gaits and tested whether limb phase values for giraffe with GSD and/or snare wounds deviated from the limb phase values of healthy giraffe.We quantified footfall patterns according to limb phase which is defined as the proportion of stride duration separating hindlimb touchdown from ipsilateral forelimb touchdown during symmetrical gaits. Limb phase patterns are associated with stability during movement51. Controlling for speed, we predicted that giraffe with snare wounds would have greater mean NSL compared to healthy individuals as a strategy to reduce loading on the affected limb41.The mean number of supporting limbs (NSL) can theoretically vary between zero  and four  throughout different portions of a stride. We quantified the portion of stride duration in which individuals were supported by zero, one, two, three or four limbs, to generate mean NSL throughout the stride52 and Vanden Hole et al.53 such that: ((Ldf − Rdf)/(0.5 (Ldf + Rdf)) × 100, where Ldf = mean of left forelimb and left hindlimb duty factors and Rdf = mean of right forelimb and right hindlimb duty factors. Values can theoretically range from − 100 to 100% with 0% indicating perfect contralateral symmetry. More negative values indicate lower duty factors on the left limbs compared to the right limbs. More positive values indicate lower duty factors on the right limbs compared to the left limbs. For animals with unilateral snare injuries, we modified the equation such that the injured/affected limb would be considered first. That is, for animals with right limb snare wounds, we used ((Rdf − Ldf)/(0.5 (Rdf + Ldf)) × 100. Thus, more negative values indicate lower duty factor on the side of the injured limb and more positive values indicate greater duty factors on the side of the injured limb. Because giraffe are typically supported by ipsilateral limb couplets for long periods of stride duration39, we predicted that individuals with unilateral snare injuries would have more negative ipsilateral DFAI values due to reduced contact time on the side of the injured limb as part of a pattern to reduce loading on that limb54. In contrast, we predicted healthy individual would have ipsilateral DFAI values equal to or close to zero .We quantified duty factor  for each of the four limbs. We then calculated an asymmetry index for ipsilateral duty factors  modified from Robinson et al.39. Basu et al.39 found that the horizontal acceleration of the neck was largely out of phase with the acceleration with the trunk which likely allows giraffe to move more efficiently . This ultimately improves the horse’s locomotor performance55. Given the importance of the neck during giraffe locomotion, we quantified neck angle during peak dorsal extension and peak ventral flexion for each stride. Points located at the base of the tail, apex of the dorsal spinous processes of the thoracic vertebrae , and apex of the occipital bone posterior to the ossicones were used to generate the neck angle. These landmarks were chosen because they were easily observable on the giraffe from our video sample  as the difference between peak ventral flexion and peak dorsal extension following Basu et al.39.A giraffe’s neck oscillates twice during a typical walking stride with peak dorsal extension  occurring during early stance phase of each forelimb and peak ventral flexion  occurring at roughly midstance of each forelimb56, including add-on packages: lme4 and lmerTest57. We used the emmeans package58 to generate estimated marginal mean values for each dependent variable listed above. Post hoc pairwise comparisons of mixed models were conducted using the emmeans package, with multiple pairwise comparisons corrected using the false discovery rate method59. Relative speed was included as a covariate in the mean NSL and neck ROM models because speed has been shown to negatively correlate with mean NSL in other taxa51 and positively correlate with neck ROM in zoo-housed giraffe39.We used linear mixed models to examine the effect of giraffe condition on spatiotemporal limb kinematics variables, including relative stride length, mean stride duration, relative speed, limb phase, mean NSL, and ipsilateral DFAI. Similarly, we used linear mixed models to assess the effect of giraffe condition on neck angle kinematics, including peak dorsal extension, peak ventral flexion, and neck ROM. For all statistical models, we included GSD (presence vs. absence) and snare wound (presence vs. absence) as fixed factors. Individual giraffe were nested within video clip as a random factor (intercept) in each model to control for random variation between individuals, providing greater power to detect meaningful variation associated with the fixed factors of interest . Satterthwaite approximations were used to adjust degrees of freedom in cases of heteroscedasticity for all models relating giraffe condition to spatiotemporal limb kinematics and neck angle kinematics. Analyses were conducted in R statistical softwareWe found that the spatiotemporal limb kinematics and neck angle kinematics of the zoo-housed Masai giraffe were generally similar to those recorded in healthy free-ranging Nubian giraffe Table . Given tWe found that GSD lesions were primarily located on the necks of giraffe in our sample Table . Snare wThe presence of GSD had a significant effect on peak ventral flexion Table ; however39. In general, all giraffe used LSLC walking gaits. The average gait cycle of the healthy Nubian giraffe recorded can be described as 66:13 in Hildebrand40 terms , which is similar to the 64:15 average gait recorded in our Cleveland Metroparks Zoo sample and the 70:14 average gait recorded by Basu et al.39. This suggests that basic kinematic parameters of giraffe walking gaits are conservative across environments and among healthy individuals of different giraffe taxa.We undertook the first in-depth investigation of the spatiotemporal limb kinematics and neck angle kinematics of walking gaits in free-ranging Nubian giraffe. We found that the spatiotemporal limb kinematics of healthy free-ranging giraffe from our sample were generally comparable to those of healthy zoo-housed Masai giraffe both from our preliminary analyses at Cleveland Metroparks Zoo and those reported in Basu et al.18, the spatiotemporal limb kinematics of Nubian giraffe with only GSD did not differ from those of healthy giraffe. This result is potentially attributable to the fact that GSD lesions were predominantly found on the necks in our sample. GSD primarily affects the necks of the MFNP’s Nubian giraffe population21, while GSD lesions are more commonly found on the limbs of giraffe taxa at other sites surveyed to date: e.g., Tanzania  and Namibia (Angolan giraffe—Etosha NP and Puros Conservancy)17. It is not yet clear why the population in MFNP is affected primarily on the neck as opposed to the limbs, although ongoing studies by Giraffe Conservation Foundation and partners are being undertaken to evaluate etiology and vector dynamics.Contrary to our predictions from the knowledge that some Masai giraffe with GSD were found to walk with greater difficulty39 found that stride frequency  was consistent across walking speeds and that giraffe increased walking speed by taking longer strides. Our results support this pattern, as giraffe with snare wounds had shorter stride lengths (but consistent stride durations) resulting in slower relative speeds. These kinematic compensations are common in animals with limb or hoof pathologies, and increased ipsilateral asymmetry, in particular, is consistent with a strategy to reduce loading on the injured limb54. The more negative ipsilateral DFAI values for giraffe with snare wounds indicate shorter contact durations, and potentially reduced peak ground reaction forces and/or impulse, on the side of the injured limb. Recording the ground reaction forces of different limbs throughout stride sequences via force plates could be used to test this assumption—a technique commonly employed to detect lameness in domestic and laboratory animals62.We found that giraffe with snare wounds had shorter stride lengths, slower walking speeds, reduced limb phase values, and more negative ipsilateral DFAI compared to healthy giraffe. Similarly, giraffe with both GSD and snare wounds had reduced limb phase values, and more negative ipsilateral DFAI compared to healthy giraffe. In their study of zoo-housed giraffe, Basu et al.63. Reduced mobility in affected giraffe may reduce foraging efficiency, limit the extent of seasonal movements, and/or reduce the amount of time giraffe can spend engaging in other behaviors  due to increased time spent locomoting. Impaired mobility may also have negative consequences on important social behaviors. For example, adult males compete for access to females with larger bulls typically out-competing other males65. Subordinate males may travel in search of other locations where female density is too high for bulls to effectively monopolize63. Locomotor deficiencies related to snare wounds and/or GSD may limit dominant males’ ability to mate guard and limit more subordinate males’ ability to travel in search of other breeding opportunities. Finally, although giraffe predation is rare in MFNP21, reduced mobility and potential flight ability may make affected individuals more vulnerable to predators. Muneza et al.27 examined relationships between GSD and lion predation in Ruaha NP—a site where giraffe are commonly preyed upon by lions. The study documented a positive relationship between severe GSD lesions and signs of attempted lion predation . This suggests lions may preferentially target individuals with severe GSD; however, the authors found no evidence that GSD lesions impacted the likelihood of surviving a lion attack and were not able to record GSD presence or severity on giraffe killed by lions.The decreased mobility and locomotor efficiency of individuals with snare wounds could have important ramifications for giraffe health and fitness. Giraffe in MFNP travel ~ 14 km per day , whilst some individuals, especially subadult and adult males, embark on seasonal migrations related to changes in food availability in the park. As part of these seasonal migrations, individuals may travel up to 30 km between acacia savanna in the wet seasons and broadleaf savanna in the dry seasons39. Basu et al.39 modelled neck accelerations and mean ground reaction forces to assess the relationship between the accelerations of the trunk and neck. They found that the horizontal acceleration of neck was largely out of phase  with the horizontal acceleration of the trunk, essentially decoupling neck movement from the rest of the body which likely results in significant energy savings. We did not find any significant differences in peak dorsal extension or peak ventral flexion of the neck when comparing animals with GSD and/or snare wounds to healthy individuals. Controlling for speed, neck ROM did not differ among the different condition categories. This is noteworthy given the prevalence of GSD lesions on the neck of giraffe in our sample and suggests that normal neck ROM during walking gaits is attainable despite GSD lesions on the neck.Walking gaits were characterized by two oscillations of the neck (one for the left limbs and one for the right limbs). Peak dorsal extension of the neck was consistent with early stance phase of each forelimb and peak ventral flexion occurred at roughly midstance of each forelimb as other have described22 found that GSD was especially common on the forelimbs  but less common on the hindlimbs of Masai giraffe in Ruaha and Serengeti NPs. This provides an opportunity to examine how forelimb vs. hindlimb, and unilateral vs. bilateral limb lesions impact gait kinematics. Researchers have previously categorized the severity of GSD by estimating lesion sizes18 or quantifying lesion sizes via photogrammetry techniques22, rather than report presence vs. absence44 as we have done here due to limitations in our sample size. Another recommended line of research would be to test whether severity of GSD  differentially impacts giraffe locomotion.Future research examining the spatiotemporal gait kinematics of the various giraffe taxa at locations where GSD is common on the limbs is required to better determine the extent to which GSD may affect locomotion. Muneza et al.44; however, future study is required to examine locomotor kinematics of giraffe at sites where lesions are found predominantly on the limbs to better assess the potential significance of GSD on giraffe locomotion and associated morbidity and mortality. We found that individuals with snare wounds showed more discernible kinematic compensations consistent with reduced speed and minimized contact on the injured limb. It is likely that some severe snare wounds impact locomotor efficiency and flight capability to the extent that they increase mortality in affected individuals. Furthermore, many animals, including giraffe, do not escape from snare entrapments30. In long-term surveys of MFNP, Mudumba et al.30 observed the highest density of wire snares recorded to date in sub-Saharan Africa and recorded the remains of fifteen giraffe caught in wire snares. Targeted veterinary de-snaring efforts in MFNP from February 2019 to December 2021 effectively removed snares from 257 live giraffe, emphasizing the severity and scope of this threat . Ongoing studies in MFNP seek to evaluate the potential fitness costs and impacts of GSD and snare wounds on survival. Locomotor kinematic studies like ours can provide crucial mechanistic perspectives on emergent patterns of larger scale movements and demographic consequences. MFNP supports the largest known population of the critically endangered Nubian giraffe remaining in the world, so identifying and quantifying potential threats to this population is of global consequence for their conservation42.Overall, we found that MFNP’s Nubian giraffe spatiotemporal limb kinematics and neck angle kinematics were largely conservative between healthy individuals and those with GSD. Our results are consistent with the idea that GSD does not appear to increase mortality of affected giraffe and does not warrant veterinary interventionSupplementary Information.