This study aims to identify heat shock protein70-2 (HSP70-2) and protamine-1 (PRM1) mRNA and protein in Madura bull sperm and demonstrate their relation as bull fertility biomarkers.The Madura bull fertility rates were grouped based on the percentage of first service conception rate (%FSCR) as high fertility (HF) , and low fertility (LF) . mRNA of HSP70-2 and PRM1 with peptidylprolyl isomerase A (PPIA) as a housekeeping gene were determined by quantitative real-time polymerase chain reaction, while enzyme-linked immunoassay was used to measure protein abundance. In the post-thawed semen samples, sperm motility, viability, acrosome integrity, and sperm DNA fragmentation index were analyzed. Data analysis was performed on the measured parameters of semen quality, relative mRNA expression, and protein abundance of HSP70-2 and PRM1, among the bulls with various fertility levels (HF and LF) in a one-way analysis of variance analysis. The Pearson correlation was used to analyze the relationship between semen quality, mRNA, proteins, and fertility rate.Relative mRNA expression and protein abundance of HSP70-2 and PRM1 were detected and were found to be highly expressed in bulls with HF (p<0.05) and were associated with several parameters of semen quality.HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility. Bos indicus and Bos javanicus [Madura cattle, which have various advantages and great potential for breeding are local Indonesian beef cattle derived from crosses of avanicus . Howeveravanicus  reportedavanicus , is not avanicus . Recent avanicus . The preavanicus .Heat shock protein70-2 (HSP70-2) and protamine-1 (PRM1) are known to play active roles in fertilization. Heat shock protein, a group of proteins with abundant chaperones, plays a role in protecting cells from apoptosis and oxidative stress . HSP70-2Fertilization being a complex process, no single test or attribute can identify and distinguish bulls as being highly fertile or infertile. Due to suboptimal sensitivity and specificity, tests diagnosing fertility based on a single feature or marker may be less effective. The trend of identifying the most important fertility markers and combining the results to improve diagnostic accuracy is gaining precedence. Therefore, the characterization of potential genes and proteins can help to comprehensively understand the molecular basis and associations between sperm quality and fertilization, further enabling the identification of these genes as potential fertility biomarkers. Therefore, in this study, we identified and analyzed mRNA and protein expression levels of HSP70-2 and PRM1 in post-thawing sperm in Madura bulls and examined their association with fertility and semen quality. The results of this study are expected to reinforce the potential of HSP70-2 and PRM1 as prospective fertility biomarkers and, can be influential in increasing the population and genetic quality of native Indonesian, Madura cattle.Frozen semen samples, collected during the dry season, which spanned from May to October from Madura bulls, maintainedble at Lembang AI Center, West Java, Indonesia, and Singosari AI Center, East Java, Indonesia were used for this study. Temperatures at these centers during semen collection ranged between 15°C to 30°C. One limitation of this study is that only eight Madura bulls from both AI Centers belonged to the productive age group (4 to 8 years old). These eight Madura bulls were classified based on the percentage of first service CR (%FSCR). The %FSCR assessment uses data from the National Animal Health Information System (iSIKHNAS), Indonesia, particularly in East Java, from 2018 to 2020. iSIKHNAS is a nationally integrated system that contains data on AI services and pregnancy examination data. It also includes data on bulls used for AI across all regions in Indonesia. Based on the data obtained from iSIKHNAS, the total number of inseminated Madura cows was 1,054. As previously reported, at least 100 insemination records are required for determining a statistically adequate %FSCR for each bull . In accoThe quality analysis was carried out on frozen-thawed sperm in this study. Assessment parameters included sperm motility, viability, the integrity of acrosome, and DNA fragmentation index (DFI). The frozen sperm straws were thawed for 30 seconds at 37°C and then were immediately analyzed to determine each parameter. Five replicate straws for each bull were used in this study. Computer assisted sperm analysis  with SpermvisionTM 3.7 programs were used to evaluate the total and progressive motility of sperm, in accordance with the method described by Sundararaman et al . Sperm mThe integrity of the sperm acrosome was analyzed using fluorescein isothiocyanate conjugated peanut agglutinin and propidium iodide . Semen samples were air-dried at room temperature, followed by fixation in 96% ethanol at room temperature for 10 minutes and air-drying. Up to 30 μL of the 100 μg/mL PNA lectin solution was poured onto the smear sample followed by incubation at 37°C for 30 minutes. The smear sample was then incubated for 5 minutes with 5 μL of 1 μg/μL PI solution , after which it was washed three times with phosphate buffer saline (PBS) to eliminate any unbound reagent residue. The slide was then covered with a cover slip, and sperm acrosome integrity was evaluated by fluorescence microscopy  using a filter at 40× magnification. Acrosome-intact sperm showed a strong green acrosomal fluorescence cap, while sperm with damaged acrosome demonstrated a red fluorescence cap .2O for 5 minutes in a dark environment. The slide was subsequently covered after washing specimen with distilled water. A total of 500 sperm cells were counted and evaluated under a fluorescent microscope  using a filter at 400× magnification. Sperm cells with a normal DNA content exhibited green fluorescence, while those with fragmented DNA content exhibited a fluorescence that ranged from yellowish-green to reddish-orange [Sperm DFI was assessed using acridine orange (AO) staining. A smear of 5 to 10 μL semen was fixed on a slide for two hours using Carnoy’s solution . The smear sample was dyed using 1% AO, 0.1 M citric acid, and 2.5 mL Nah-orange . The res−ΔΔCT technique was used for quantifying the qRT-PCR expression data for each gene [Direct-zol RNA Miniprep Plus separated total RNA from post-thawed sperm . The sperm RNA solution was stored in the RNase-free tube at −80°C. RNA quality and quantity was assessed using Nanodrop . RNA samples with a 260/280 ratio of 1.8 to 2.0 were considered for cDNA synthesis. The cDNA was synthesized in accordance with the manufacturer’s protocol using the SensiFast cDNA synthesis kit (Meridian Bioscience (Bioline), Memphis, TN, USA). The cDNA synthesis procedure was carried out by adding several reactions with a total volume of 20 μL. The total volume of RNA used was 10 μL. The cDNA samples thus obtained were used for quantitative real-time polymerase chain reaction (qRT-PCR), analysis. The Sensifast SYBR mix  was used for the qRT-PCR analysis. The compositions used for the reactions included 1 μL each of forward and reversed primer, 10 μL of Sensifast SYBR mix, 6 μL of nuclease-free water, and 2 μL of cDNA stock sample. The qRT-PCR protocol was performed using CFX Opus 96  with an activation step of 95°C for 2 min. Amplification was carried out at a denaturation temperature of 95°C for 10 s for 40 cycles, annealing temperature according to the primer of each gene  for 30 sach gene .6 sperm cells/mL). The amount of HSP70-2 and PRM1 protein in the sperm pellets were then measured using the enzyme-linked immunoassay (ELISA) method, in accordance with the manufacturer’s instructions . Briefly, after preparation of the reagents, samples, and standards, the sperm pellets obtained after centrifugation were placed into the wells. The sperm pellet samples and each standard of bovine HSP70-2 and PRM1 were added to the respective reaction wells, which were then covered with adhesive tape and incubated at 37°C for 90 minutes. The anti-bovine HSP70-2 and PRM1 biotinylated antibody solutions were prepared 30 minutes before conducting the experiment. The EIA plate was washed twice, and 100 μL of antibody solution was added to the wells, which were then sealed with adhesive tape and incubated for 60 min at 37°C. The EIA plate was rinsed three times followed by the addition of 100 μL of an enzyme-conjugated solution that had been prepared 30 minutes earlier. The wells were then sealed and incubated at 37°C for 30 min. The EIA plate was then rinsed five times, the Color Reagent solution (100 μL) was added, and then the plate was incubated at 37°C for 30 minutes in the dark. In the final stage, 100 μL of Color Reagent C was added and was thoroughly mixed. In the end, an EIA reader was used to measure the number of HSP70-2 and PRM1 proteins in sperm with absorbance at 450 nm within 10 minutes.Each frozen sperm sample was thawed in a 37°C water bath for 30 seconds, washed with PBS and then centrifuged at 700×g for 15 minutes to pelletize it  based on the Kolmogorov–Smirnov test. Data analysis was performed on the measured parameters of semen quality, relative mRNA expression, and protein abundance of HSP70-2 and PRM1 among the bulls with various fertility levels (HF and LF) in a one-way analysis of variance analysis. In the event of a significant difference in each fertility group, Tukey’s post hoc test was applied. Pearson correlation was used for analyzing the relationship between semen quality, mRNA, proteins, and fertility rate. A linearity test was performed using Scatter Plot, for analyzing the relationship pattern between HSP70-2 and PRM1 mRNA expression and protein abundance with fertility rate (%FSCR). The data is presented in the form of mean±standard error. Statistical analysis was performed using statistical package for social sciences version 26 .The results of the quality analysis of the semen samples obtained from HF and LF Madura bulls are presented in The mean percentages of acrosome-intact sperms were 83.1%±4.1% and 72.1%±6.7% in the HF and LF bulls respectively. The HF bulls had more acrosome-intact sperm than LF bulls (p<0.05). The mean percentage of sperm DFI in HF and LF bulls was 14.6%±1.3% and 17.2%±0.8%, respectively. However, LF bulls had a larger percentage of DFI (p<0.05) than HF bulls.The results indicated that semen quality parameters were associated with fertility rates and were negatively correlated with %DFI . Sperm mThe relative mRNA expression and protein abundance of HSP70 and PRM1 in each fertility level has been depicted in mRNA expression of HSP70-2 positively influenced the fertility levels (p<0.01), sperm total (p<0.01) and progressive (p<0.05) motility, viability (p<0.05), and acrosome integrity (p<0.01). Similarly, PRM1 mRNA expression also positively influenced the fertility levels (p<0.01), sperm total (p<0.05) and progressive (p<0.01) motility as well as acrosome integrity (p<0.05). At the protein level, HSP70-2 had a positive correlation with fertility levels (p<0.01), sperm total (p<0.01), and progressive (p < 0.05) motility. PRM1 protein abundance, on the other hand, had a positive correlation with fertility levels (p<0.01), sperm total motility (p<0.05), viability (p< 0.05), and acrosome integrity (p<0.01). HSP70-2 mRNA expression and its protein abundance as well as PRM1 mRNA expression and its protein abundance were found to be with DFI p<0.05; .The results of the linearity test are presented in Fertility of bulls is one of the determining factors for the success of AI programs. Semen quality plays an important role in determining the success of AI . Frozen During analysis of sperm viability, the percentage of damaged sperm membranes was found to be higher in LF bulls than in HF bulls, indicating reduced resistance of sperm to external factors in LF bulls. Acrosome integrity was also significantly lower (p<0.05) in LF bulls, thereby demonstrating a close relationship (p<0.01) between acrosome integrity and fertility. This is in agreement with a report by Pardede et al , accordiOverall, even though the semen quality parameters in this study were within a reasonable acceptable range and were classified as “good,” and had even met the specified requirements , it was evident that this was not sufficient in assessing the fertility level of bulls. According to a previous report by Butler et al , conventHeat shock protein member 2 and PRM1 are known to play an active role in fertilization ,21,22. HHeat shock protein70-2 is a molecule that is adjacent to ubiquitin in sperms. It is involved in spermatogenesis, protein binding, metabolism, and catalytic activity related to sperm function, and hence is critical in the process of fertilization. It not only impacts the success of fertilization, but also plays a role in the early development of the embryo . Meanwhi2+-ATPase activity, reducing membrane damage, and increasing mitochondrial function. In addition, HSP70 stimulates superoxide dismutase activity and protects sperm from further oxidative damage. The HSP family is also reported to have a higher cellular tolerance to various types of stress that might have a damaging effect on cells. Thus, the HSP70-2 molecule protects mitochondria and intracellular sperm from stressors and is associated with the normal function of sperm motility in bulls [In general, HSP70-2 functions by increasing Cain bulls . Chen etin bulls  have prein bulls  that, pain bulls  also repin bulls . These f2+ in sperms with PRM deficiency results in the degradation of the plasma membrane, reduced sperm motility, and greater DNA damage. A high %DFI was closely related to low mRNA expression, and an abundance of PRM1 protein (p<0.05) (mRNA expression and protein abundance of PRM1 were significantly higher (p<0.05) in HF bulls  and is s(p<0.05) . As the (p<0.05) . Accordi(p<0.05) . The posin vitro, is needed to further validate the results of this research. A study of the potential of HSP70-2 and PRM1 using bulls of different age groups, as well as productive and non-productive bulls, mainly related to standardization and determination of the abundance of HSP70-2 and PRM1 contained in frozen-thawed sperm needed by a bull to be categorized as a bull with HF will further strengthen the results of this research. However, this study supports the categorization of HSP70-2 and PRM1 as two of the many genes or proteins in sperms, that have great potential to be developed as biomarkers to accurately assess the fertility of bulls.Nonetheless, further studies are warranted to further strengthen this hypothesis and the findings of this study regarding the potential of HSP70-2 and PRM1 as potential biomarkers for predicting bull fertility. Research using a similar experimental design and carried out on other bull breeds will significantly enrich the results of this study. Considering that this research was carried out only on Madura bull sperm, anticipation of the influence of breed factors on the research results would be necessary. However, a biomarker would be expected to be applicable to all bull breeds and not just one bull breed. In addition, investigation on the association of HSP70-2 and PRM1 on the ability of sperm-oocyte fertilization, especially In this study, we have revealed the association between mRNA expression and protein abundance of HSP70-2 and PRM1 in varying fertilities of Madura bulls as indigenous cattle in Indonesia. Relative mRNA expression and protein abundance of HSP70-2 and PRM1 were identified and were found to be abundant in bulls with HF and were associated with several parameters of semen quality. Each parameter of semen quality was seen to exist at a lower percentage in bulls with low fertility in this study. Semen quality parameters have a close relationship with the level of fertility. HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility.


Influenza viruses, especially Influenza A virus and Influenza B virus, are respiratory pathogens and can cause seasonal epidemics and pandemics. Severe influenza viruses infection induces strong host-defense response and excessive inflammatory response, resulting in acute lung damage, multiple organ failure and high mortality. Isoquercitrin is a Chinese medicine monomer, which was reported to have multiple biological activities, including antiviral activity against HSV, IAV, SARS-CoV-2 and so on. Aims of this study were to assess the in vitro anti-IAV and anti-IBV activity, evaluate the in vivo protective efficacy against lethal infection of the influenza virus and searched for the more optimal method of drug administration of isoquercitrin.In vitro infection model (MDCK and A549 cells) and mouse lethal infection model of Influenza A virus and Influenza B virus were used to evaluate the antiviral activity of isoquercitrin.Isoquercitrin could significantly suppress the replication in vitro and in vivo and reduced the mortality of mouse lethal infection models. Compared with virus infection group, isoquercitrin mitigated lung and multiple organ damage. Moreover, isoquercitrin blocked hyperproduction of cytokines induced by virus infection via inactivating NF-κB signaling. Among these routes of isoquercitrin administration, intramuscular injection is a better drug delivery method.Isoquercitrin is a potential Chinese medicine monomer Against Influenza A Virus and Influenza B Virus infection.The online version contains supplementary material available at 10.1186/s13020-023-00843-4. Orthomyxoviridae family, is a single-stranded, negative sense RNA virus and is one of the most significant pathogens threatening to human health. Influenza viruses are classified into four types , of which influenza A virus (IAV) comprised the majority of global seasonal influenza case. Co-circulating with IAV, influenza B virus (IBV) is also responsible for about 25% of all influenza-related hospitalisations, especially cause high morbidity and mortality in children. [Influenza virus, belonging to the hildren. , 2. ContIsoquercitrin, also known as Quercetin-3-O-glucoside, is a kind of flavonol and widely exists in plants, like Houttuynia cordata, Hedyotis diffusa, et al. Currently, besides the direct extraction from plants, the chemical synthesis method was extensively used to prepare isoquercitrin. As a mono-glycoside derivative of quercetin which has diverse biological functions, isoquercitrin has been demonstrated to exhibit anti-tumor , anti-apIn this study, we found that isoquercitrin exhibited suppressive effect on influenza A (H1N1) virus and influenza B virus in vitro. Furthermore, isoquercitrin possesses a therapeutic effect in animal models through enhancing virus clearance, suppressing inflammation and attenuating lung pathological injury. Additionally, compared with other drug administrations, intramuscular injection is a better drug delivery method. These findings suggested that isoquercitrin might be a potential antiviral candidate against influenza A virus and influenza B virus.Human lung adenocarcinoma A549 cells and Madin-Darby canine kidney cells (MDCK) –21 were 4 cells/well) and incubated at 37 °C with 5% CO2. When the confluence reached 70–80%, different concentrations of the drug  were added. After 48 h, cell viability was measured by CCK-8 assay using Cell Counting Kit-8  following manufactory’s instruction. For in vitro antiviral assay, cells were washed with PBS and infected with influenza A virus (H1N1/PR8 and H1N1/UI182) and influenza B virus (IBV/S9-E2 and IBV/S9-MD) (MOI = 0.1) when cell confluence reached 70–80%. After 1 h post infection, isoquercitrin were added. After 48 h, cell viability was measured by CCK-8 assay. As for time-of-addition assay, cells were treated with isoquercitrin for 1 h before virus infection or inoculated with isoquercitrin-viruses mixture, after 1 h virus adsorption. At 48 h post-infection, cell viability was measured by CCK-8 assay.MDCK and A549 cells were seeded in 96-well plate (10® 488), Abcam, ab150077, 1:500) for 2 h without light. After nuclei staining with Hoechst 33,258  for 10 min, fluorescence was observed under a fluorescence microscope .The cells were seeded in 12-well plates and infected with H1N1/UI182 (MOI = 0.5) when confluence reached 70%. At 48 h post-infection, cells were washed three times with cold PBS and fixed with 4% paraformaldehyde (PFA) for 20 min, then the plate was infiltrated with 0.2% Triton-X100 and blocked with 2% BSA for 1 h. The primary antibody against influenza virus Nucleocapsid protein  was added and incubated with cells overnight at 4 ℃. After the primary antibodies, cells were washed three times using PBS and incubated with the secondary antibodies (Goat Anti-Rabbit IgG H&L (Alexa Fluor50) using nasal drop method, respectively. Control group and virus-infected groups were treated with 0.9% saline by intraperitoneal injection (i.p.). The isoquercitrin treatment  [Female BALB/c mice  were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd . After 3 days of adaptive feeding, mice were randomly divided into uninfected group (Control), virus-infected groups (H1N1/PR8 and IBV/S9-MD), isoquercitrin-treated group, oseltamivir phosphate (a NA inhibitor)-treated group. Mice in each group, except uninfected group, were challenged with 50 µL virus dilution   and oselay, i.p)  started At 3 day post-infection (3 dpi) and 5 day post-infection (5 dpi), lung, heart, liver, spleen and kidney of mice in each group were collected, part of tissue was used for analysis of gene and protein expression, part of tissue was fixed in the 4% paraformaldehyde (PFA), preparing for tissue sections. For pathological analysis, tissue sections were stained with hematoxylin and eosin (H&E), dried and observed under a light microscope . Immunohistochemical assay was performed as described previously .™ RT reagent Kit . The expression level of target mRNA was measured by qRT-PCR assay using TB Green Premix Ex Taq II  based on TB Green chimeric fluorescence method. The primer pairs were listed in Table β-actin was used as a loading control.Total RNA from mouse lung tissues of mice was extracted using the HiPure Universal RNA Kit Magen, R4130-03). About 1 μg RNA was used to synthesize cDNA using PrimeScript30-03. Ab50 (50% egg-infective dose) of virus. An equivalent number of mouse lung tissues from each group were crushed in DMEM supplemented with 400 units/mL penicillin and streptomycin. Followed by centrifugation, the supernatant was serially diluted tenfold from 10–1 to 10–8 and were inoculated into eggs for 48 h at 37 ℃ . Then 50 μL allantoic fluid in each egg was collected and mixed with 50 μL 1% chicken RBC. After standing for 15 min, the highest dilutions that completely agglutinated chicken RBC were recorded. Viral titer was calculated by the Reed-Muench method, and the result was expressed as log10 EID50.9 day-old embryonated chicken eggs were used to detect EIDThe mouse lung tissues from each group were lysed with RIPA Lysis Buffer  to collect total proteins. After protein quantification and electrophoresis, the proteins were transferred on a PVDF membrane. The protein expression was detected by relative antibody and β-actin  was used as an internal reference protein for quantitative analysis.Statistical comparisons were performed using ANOVA analysis. Quantitative data sets were expressed as the means ± standard error (SE), and the statistical significance was evaluated by Graphpad Prism 8.0 software. Compared with the Virus-infected group, P < 0.05 was regarded to have significant differences.*P < 0.05, **P < 0.01, and ***P < 0.001.50 > 800 μg/mL), with EC50 of 46.77 ± 1.31 μg/mL for H1N1/PR8  higher than 10 . To investigate which stage of influenza virus infection does isoquercitrin exert the antiviral effect, time-of-addition assay was performed. The inhibitory effect of different drug administration protocols showed that isoquercitrin played an antiviral role main in late stage of influenza virus infection, as treatment after infection exerted the maximum antiviral effect against both IAV and IBV  and influenza B virus (IBV/S9-MD). After corresponding treatment, four mice of each group were euthanized and tissues  were collected for further analysis. The remained mice are used to monitor the body weight change and survival status. The results showed that virus infection induced obvious reduction of mouse body weight from day 2 and all mice in H1N1/PR8-infected group and IBV/S9-MD-infected group dead by day 6 after infection. In contrast, isoquercitrin and oseltamivir treatment attenuated weight loss and increased the survival rate of infected mice. Surprisingly, isoquercitrin significantly promoted mouse body weight recovery, which was better than oseltamivir, but its protective effect was inferior to Oseltamivir Fig. C, D and Influenza virus infection can cause variable degree of lung injury in host which is an important standard of therapeutic efficacy of antiviral drugs . Thus, wIFN-α , intramuscular injection (i.m.), oral gavage (p.o.), and subcutaneous injection (s.c.). The results displayed in Fig. 50 values for influenza A virus were in the low nanomolar (~ 2–13 nM) concentration range [IAV infection is main causes of seasonal influenza epidemics and pandemics which can induce human respiratory disease and acute lung injury (ALI). Meanwhile, IBV often co-circulates with IAV and even becomes major epidemic strains worldwide . Existinon range , but in Ifnar1−/− mice [Isoquercitrin is a flavonoid that can be extracted from natural plants and exhibits antiviral activity against multiple viruses. Isoquercitrin was reported to be the main component of some Traditional Chinese medicine (TCM) that suppressed HSV by inhibiting ROS production and NF-κB activation , 10, sup−/− mice , 35. TakCytokine is a kind of peptide or glycoprotein secreted by the cells and perform diverse biological functions, such as immune modulation, involvement in inflammatory responses, etc. Cytokine is a crucial component of antiviral innate immune, but severely viral infections are always accompanied by cytokine dysregulation and amplification, even becomes the main factor for viral pneumonia during the later phase after virus infection . ConsideThe pathogenicity of an influenza virus depends on many factors, such as cell tropism, replicate efficiency, and host immunity. Both IAV and IBV prefer to replicate in respiratory system and innate immune cells are recruited to lung and produce massive cytokines and chemokines after influenza virus infection, inducing pneumonia, even multi-organ failure . UnexpecThe dose and route of administration are main factors affecting the drug efficacy . In otheAbove findings suggest that isoquercitrin has potent antiviral activity against influenza A (H1N1) virus and influenza B virus in vitro and in vivo. Lethal infection of influenza virus induced excessive production of cytokines and severe lung damage, but these outcomes were suppressed by isoquercitrin, indicating that this drug could benefit severe patients with influenza virus infection. Additionally, through comparison between different drug delivery methods, the i.m. was proved as a better method for administration of isoquercitrin.Additional file 1: Fig. S1. The effect of timing of drug administration and dose on anti-IAV A–B and anti-IBV C–D activity of isoquercitrin in MDCK cells and A549 cells. Colors annotate three drug administration protocols, pre-treatment (gray), co-treatment (black) and post-treatment (pink). Fig. S2. Effect of isoquercitrin on pathological damage caused by influenza virus infection. A HE staining of heart, liver, spleen and kidney tissues in influenza virus-infected mice and drug-treated mice at 5 dpi. B Immunohistochemistry staining of heart, liver, spleen and kidney tissues in influenza virus-infected mice and drug-treated mice at 5 dpi. C The percentage of NP positive cells in main tissues  in B. Fig. S3. Isoquercitrin inhibits cytokines induced by influenza A virus infection. Relative mRNA expression level of type I IFN (IFN-α and IFN-β), pro-inflammatory cytokines , anti-inflammatory cytokine (IL-10) and IFN induced gene (ISG54 and ISG56) in mouse lung tissues of each group at 5 dpi.


Exchange spring magnetic (ESM) behavior between LSMO and LMCO, manifested by a spin reorientation transition of the LSMO layers towards perpendicular magnetic anisotropy below TSR = 260 K, was observed. Further, 3ω measurements of the [(LMCO)9/(LSMO)9]11/STO(100) superlattices revealed extremely low values of the cross-plane thermal conductivity κ(300 K) = 0.32 Wm−1K−1. Additionally, the thermal conductivity shows a peculiar dependence on the applied IP magnetic field, either decreasing or increasing in accordance with the magnetic disorder induced by ESM. Furthermore, both positive and negative magnetoresistance were observed in the SL in the respective temperature regions due to the formation of 90°-Néel domain walls within the ESM, when applying IP magnetic fields. The results are discussed in the framework of electronic contribution to thermal conductivity originating from the LSMO layers.Superlattices (SLs) comprising layers of a soft ferromagnetic metal La Their hallmark is a strong coupling between charge, spin, and lattice degrees of freedom, which results in super- and double-exchange (SE and DE) mechanisms of orbital interactions /κ(B = 0), can be observed in magnetoresistive materials due to the variation of their electrical conductivity dependent on a magnetic field m SLs epitaxially grown on STO(100) substrates via a metalorganic aerosol deposition (MAD) technique (see Methods section for details). Such an exchange spring with PMA, dictated by the hard magnetic LMCO layers, strongly impacts the electrical and thermal transport properties of SLs through magnetic field control of spin order/disorder. The results obtained highlight an attractive opportunity to study the spin-dependent scattering of charge carriers at domain-wall-hosting interfaces as well as to control the electromagnetic and thermal properties of SLs with a magnetic field.Here we report an ESM coupling between LSMO and LMCO within m/STO(100) SLs were grown with an overall thickness of around d ≈ 75 nm by varying the superlattice repetition number “m”.All oxide film samples were prepared by means of the metalorganic aerosol deposition (MAD) technique. Aerosols of the metalorganic precursor solution  have been sprayed through compressed air on a heated substrate. By using precise dosing units for liquid precursors as well as in situ growth control by means of optical ellipsometry , the sinrocedure  using an2 to fit the holder).Four-probe electrical conductivity and magnetization measurements were carried out by Quantum Design GmbH  PPMS and SQUID magnetometers MPMS XL and MPMS 3. The latter has been used for the rotator measurements (Standard rotator sample holder with α = 0° corresponding to the in-plane direction (α = 90° out-of-plane), samples were cut to 3 × 3 mm2O3) were used.The heater (150 nm thick Au layer grown on 5 nm thick Cr adhesion layer) for thermal conductivity measurements by the 3ω method was deposited by thermal evaporation (Cr) and magnetron sputtering (Au) followed by structuring with an optical lithography lift-off  exposure unit, Allresist AR-P 5350 photoresist . The dimensions of the heater line  were measured by optical microscopy and additionally confirmed by scanning electron microscopy. To electrically decouple the metal heater from the analyzed and potentially conductive samples, insulating capping layers of amorphous alumina (am-Al2O3 capping layers ρam-Al2O3 = 3.3(2) g·cm−3 from single film measurements after fitting with the GenX program m/STO(100), with layer thicknesses ranging from n = 1–24 unit cells (u.c.), were grown with SL repetition numbers m between 4 and 96 to ensure an overall thickness of d ≈ 75 nm for all samples. The structure and microstructure of the representative SLs with n = 9, 24 u.c. and m = 11, 4 are shown in q ≈ 0.2 nm for the n = 9 u.c. SL and Sq ≈ 0.5 nm for the n = 24 u.c. SL, determined at the SL surfaces by means of atomic force microscopy (c ≈ 0.3849–0.3855 nm for both LMCO and LSMO layers (see 0.7Sr0.3) as well as very similar atomic masses of B-site cations, i.e., Co(59) and Mn(54). This makes the analysis of interfacial sharpness and intermixing at the atomic scale difficult. The additionally performed TEM chemical analysis of the [(LMCO)9/(LSMO)9]11/STO(100) SL by using energy dispersive X-ray microanalysis (EDX) croscopy  additionyers see  This is EDX) see  has reven/(LSMO)n]m/STO(100) SLs, we first introduce single films of LSMO/STO(100) and LMCO/STO(100). The optimally doped perovskite manganite La0.7Sr0.3MnO3 (LSMO) is a well-known soft FM metal with an in-plane (IP) magnetic easy axis and magnetotransport properties governed by the DE interaction m/STO(100) heterostructures, by Zhang et al. m/STO(100) SLs possess a perovskite/perovskite heteroepitaxy without any structural symmetry mismatch at the interface as evidenced by high-resolution HAADF-STEM and iDPC-TEM measurements -coupled heterostructure. The reduction of the IP magnetization of our SLs below coupling . As one troscopy . Thus, tDue to the orthogonality of the easy axes of the LSMO and LMCO layers, the formation of 90° Néel-type domain walls at the interfaces between the layers at low temperatures is favored. Such ESM made from the insulating LMCO and metallic/magnetoresistive LSMO counterparts should certainly give rise to a unique field-dependent electrical resistivity behavior. Temperature- and field-dependent electrical resistance measurements  of the n = 9 u.c. SL are shown in TMI ≈ 320 K for B = 0 T and a maximal value of CMR(300 K) = −36%. This can be attributed solely to the LSMO, as current flow takes place only within the metallic LSMO. A similar R(T) behavior is known for single LSMO/STO(100) films of comparable thickness and is reported elsewhere 11/STO(100) SL is presented in κSL(300 K) = 0.32 Wm−1K−1 has been obtained in this SL sample in zero field. Similar values were reported in the literature for bulk samples of all-inorganic vacancy-ordered double perovskites  = 0.29 Wm−1K−1 (ref. 11/STO(100) SL shown in SL-LMCO ≈ 154° which is similar to bulk, while within the LSMO layers, the values reach φSL-LSMO ≈ 160°, having a stronger tilt compared to the bulk LSMO with φLSMO ≈ 166° . In betTC) and electric (TMI) transition temperatures towards higher values, which is common for perovskite FM manganites [MTC ≈ −11%. For stronger fields of 4 T < B < 9 T, the amplitude of the negative MTC decreases and even almost changes sign for B = 9 T. A possible explanation for the observed behavior is the field-induced spin disorder within the LSMO layers promoted by the ESM formation. The spin disorder affects the charge carrier mobility due to enhanced spin scattering  A snganites . Howeverring cf. d and cou2/3Sr1/3MnO3/La2CoMnO6 (LSMO/LMCO) were epitaxially grown on SrTiO3(100) substrates using the metalorganic aerosol deposition technique. Their magnetic behavior was shown to be governed by perpendicular magnetic anisotropy, originating from magnetically hard double perovskite LMCO layers. An exchange-spring magnetic coupling between LSMO and LMCO layers was observed, leading to a spin reorientation transition of LSMO at TSR = 260 K. Applied in-plane magnetic fields enable control of the degree of spin disorder through continuous spin rotation within the soft magnetic LSMO layers towards their natural in-plane easy axis. Such field-controlled spin order/disorder was found to strongly influence both electrical and thermal transport in the SLs via spin-dependent scattering of charge carriers and their contributions to thermal transport in LSMO. The results obtained highlight the suitability of exchange-spring magnetic coupling within perovskite superlattices not only for achieving perpendicular magnetic anisotropy with nanoscale layer thicknesses down to ~9 u.c. (~3.5 nm) but also for controlling both electrical resistance and thermal transport using magnetic fields.Superlattices of La


African swine fever (ASF) causes serious economic losses to the global pig industry. Unfortunately, there is currently no effective and safe vaccine, although its development is actively continuing. To date, experimental vaccines based on the use of live attenuated strains of the ASF virus (ASFV) obtained by the deletion of viral genes responsible for virulence are the most effective. However, deletion of the same gene in different ASFV isolates can lead to different results. In our work, we have shown that, unlike some other isolates, the deletion of the CD2 gene from the genome of the virulent Congo strain does not lead to its weakening, although it affects the onset of ASF infection in domestic pigs.African swine fever (ASF) is an infectious disease that causes the most significant losses to the pig industry. One of the effective methods for combating this disease could be the development of vaccines. To date, experimental vaccines based on the use of live attenuated strains of the ASF virus (ASFV) obtained by the deletion of viral genes responsible for virulence are the most effective. Deletion of the EP402R gene encoding a CD2-like protein led to the attenuation of various strains of the ASFV, although the degree of attenuation varies among different isolates. Here we have shown that the deletion of the EP402R gene from the genome of a high-virulent Congo isolate did not change either the virulence of the virus or its ability to replicate in the swine macrophage cell cultures in vitro. However, in vivo, animals infected with ΔCongo-v_CD2v had a delay in the onset of the disease and viremia compared to animals infected with the parental strain. Thus, deletion of the CD2 gene in different isolates of the ASFV has a different effect on the virulence of the virus, depending on its genetic background. African swine fever (ASF) is a viral hemorrhagic disease of domestic pigs, of which the mortality rate reaches 100% ,3. AfterAsfarviridae family [ASFV is a large DNA-containing cytoplasmic virus that belongs to the e family . Genotype family ,7. ASFV e family . Underste family .The CD2v protein of the ASFV  is a hemagglutinin, which is responsible for the characteristic formation of erythrocyte rosettes around ASFV-infected cells; it also contributes to the development of erythrocyte-associated viremia associated with the formation of protective immunity ,10,11,12We have recently shown that CD2v proteins (encoded by the EP402R gene) and/or C-type lectin (encoded by the EP153R gene) of the ASF virus are necessary to determine serological specificity in the hemadsorption inhibition reaction (HAI), and may be important for protecting pigs from infection with homologous ASFV ,19,20. PA pair of virulent Congo-v strain (K49) and attenuated Congo-a strain KK262) is used by us as a well-characterized model for studying both the functions of ASFV genes and the protective immune response against ASF ,24. Both2 is usedPreviously, we demonstrated that the deletion of CD2v in the attenuated Congo-a strain did not affect the ability of this mutant to replicate in cell cultures; however, immunization of animals with this virus did not lead to the formation of protective immunity in response to infection with the homologous virulent ASFV Congo-v strain . In thisSus scrofa domesticus) [6 cells/well) or 48-well plates (4 × 106 cells/well)  in media containing RPMI 1640 with the addition of 10% (v/v) fetal bovine serum (Gibco), 30% (v/v) plasma, and antibiotic (Gibco) in a CO2 incubator  for 24 h. The adherent cells were washed with the same medium and used in experiments after 36–48 h.A4C2/9 k cells (esticus)  and ASF esticus) . A4C2/9kesticus) . PrimaryViruses were titrated in 96-well plates based on the presence of cytopathic effect (CPE) in primary cultures of porcine macrophages. Titers were expressed as the average infectious dose of tissue culture (TCID50) using the Reed–Munch method .2). The cell suspension was collected at 0, 24, 48, 72 and 96 h after infection (hpi) and stored at a temperature of −50 °C. These viral preparations were then used to determine TCID50/mL titers in primary cultures of porcine macrophage cells.The growth curves of the parental Congo-v virus and its mutant ΔCongo-v_CD2v were measured after the infection of primary cultures of porcine macrophages in 48-well plates. Cultures were infected with viruses at MOI = 0.1 or 1; then, after 1 h of adsorption, the virus was removed; the cells were washed with PBS and incubated in a macrophage medium , using a recombination cassette consisting of the reporter gene GFP , flanked by two recombination arms, as described earlier ,20. A4C2The recombinant virus was selected by the method of limited dilutions in primary cultures of porcine macrophages using fluorescence microscopy. The purity of the selected virus was confirmed using real-time PCR with primers specific to the EP402R gene or to the recombination site as described earlier . The seqThe absence of CD2v gene expression in cells infected with ΔCongo-v_CD2v was confirmed in hemadsorption assay (HA) according to the standard protocol . In shorAll animal procedures were conducted in accordance with Russian legislation on the protection of animals used for scientific purposes, and the experiments were approved by the Research Ethics Committee of the Federal Research Center of Virology and Microbiology, Russia .3 TCID50. Pigs from group 3 (n = 5) were left as controls and were inoculated with a PBS buffer.Piglets not vaccinated against any infections were received from a commercial pig farm (Russia). A total of 18 white pigs  aged 2–2.5 months (weighing 15–18 kg) were obtained. The piglets were randomly divided into three groups. Each group of animals was kept in isolated rooms throughout the experiment. After a 1-week acclimatization period, pigs from group 1 (n = 5) were infected intramuscularly with Congo-v virus. Pigs from group 2 (n = 8) were also infected intramuscularly with the ΔCongo-v_CD2v virus. Both viruses were injected at a dose of 10The pigs’ rectal body temperature and clinical signs were measured daily throughout the experiment. Clinical evaluation of ASF was performed in 4 different categories . The clinical signs were assigned numerical values based on the severity and significance as described in . The totTime to death and survival were recorded as described earlier . The dayThe viral load in the blood and organ samples of infected animals was assessed using titration in the primary porcine macrophage cell culture, as described above.To study the function of CD2v in virulent variants of the ASF virus, a recombinant Congo virus (Congo-v) with deletion of the EP402R gene was constructed. This ΔCongo-v_CD2v virus was obtained by homologous recombination using a construct containing two “arms” of recombination and a GFP reporter gene under the ASFV promoter p72 A. The reThe recombinant virus was obtained as a result of selection during nine rounds of limiting dilutions in primary cultures of pig macrophages. The absence of a wild-type virus, as well as the presence of a recombination event in the final preparation of the recombinant virus, was confirmed using PCR. Real-time PCR with primers specific to the EP402R gene showed the absence of the target product. PCR analysis with primers flanking the recombination site revealed an expected fragment of 2.35 kb in size in the case of the parent Congo-v virus and a fragment of 2.15 kb in size in the case of the recombinant ΔCongo-v_CD2v virus. The replacement of the EP402R gene with the GFP gene and the absence of additional mutations in the fragment ΔCongo-v_CD2v genome were also confirmed by sequencing.Hemadsorption in macrophage cell cultures infected with recombinant ΔCongo-v_CD2v was not observed, which also confirmed the absence of CD2 protein due to the deletion of the EP402R gene C. MoreovTo study the role of the CD2v protein in the replication of ASFV, in vitro multistage growth curves of viruses in primary cultures of pig macrophages were constructed. The growth curves of the ASF virus ΔCongo-v_CD2v and the parental virulent ASF virus Congo-v were measured in cells infected with viruses with an MOI equal to 1 or 0.1. The multiplicity of infection equal to 0.1 was used to ensure multiple replication cycles during the experiment. Infected cells were used to determine TCID50/mL titers in primary cultures of swine macrophage cells. The analysis of the data obtained showed that no differences in the growth of viruses were detected until the end of the observation period (96 hpi) . In addi3 TCID50. Pigs from group 3 were inoculated with PBS and were used as a control in the experiment. Animal observations showed that all control pigs had no clinical signs associated with the disease  were comparable in two groups B. Due toA summary of the animal experiment is given in African swine fever causes huge economic losses to the pig industry all over the world. To date, no effective and safe vaccines have been developed, but the greatest success has been achieved with the use of live attenuated viruses. These non-pathogenic viruses can be identified in nature, as well as obtained in the laboratory by deletion of the genes responsible for virulence.Along with the genes I177L, 9GL, UK, I226R, A137R and MGF360/MGF505 ,36,37,38It was shown that deletion of CD2v from the virulent strain BA71 (genotype I) or the strain ASFV-Kenya-IX-1033 (genotype IX) led to the virus attenuation, and these mutants induce protective immunity against infection with a parental or heterologous virus ,21. In aTo assess the function of CD2v on the replication of virulent ASFV isolates in cell cultures and in domestic pigs, a recombinant virus ΔCongo-v_CD2v containing the deletion of the EP402R gene was obtained. As we have previously shown, a pair of ASFV Congo strains (virulent K49 and attenuated KK262) is a promising model for studying functional genomics and protective immunity against ASF ,24. AnalInterestingly, our data are consistent with the data obtained in the experiment involving the Malawi Lil-20/1 isolate (genotype VIII) . CD2v deHowever, there are examples of pathogenic non-hemadsorbing ASFV isolates . In addiAn attempt was made to improve the experimental vaccine strain ASFV-G-Δ9GL by additional deletion of the EP402R gene . It is iContradictory data on the effect of CD2 protein on the virulence of various strains may be due to the fact that other ASFV genes can functionally compensate for the loss of function of the EP402R gene. However, it is currently not known which ASFV genes may be involved in this mechanism. One of the candidates for this role may be the genes of multigene families (MGF). The essential roles of MGF proteins in in vivo infections, virulence and immune escape have been demonstrated repeatedly ,48,49,50The results presented here confirm that direct extrapolation of the results obtained as a result of deletions of individual genes in a particular strain of the virus to other viral isolates should be avoided. Moreover, the result of genetic modification of the ASF virus can be unpredictable, and in each case, it requires careful experimental verification.The EP402R gene (encoding the CD-like protein) is one of the genes whose deletion can lead to a reduction in the virulence of the ASF virus. The CD2v protein is responsible for mediating hemadsorption and participates in the formation of a protective immune response. Deletion of the EP402R gene led to the attenuation of various strains of the ASFV, although the degree of attenuation varies among different isolates. In this paper, we have shown that the deletion of the EP402R gene from the genome of a high-virulent Congo isolate did not change either the virulence of the virus or its ability to replicate in the swine macrophage cell cultures in vitro. However, the onset and the mean value of viremia in animals infected with ΔCongo-v_CD2v were significantly delayed. Thus, deletion of the CD2 gene in different isolates of the ASFV has a different effect on the virulence of the virus, depending on its genetic background.


To determine severe sepsis (SS) incidence, hospital mortality, 1-year mortality, and costs associated with care in a sample of enrollees in a nationally representative individual practice association (IPA)-network managed care organization (MCO).This was a retrospective analysis of administrative claims data for commercial (not managed Medicare) members. We identified MCO members hospitalized for SS between July 1995 and December 1998. SS cases were identified by a combination of ICD-9-CM codes for infection and organ dysfunction. Enrollment information, physician, facility, and pharmacy claims were analyzed. Subjects with continuous enrollment were followed for 1 full year of observation. Costs were health plan payments to providers, after subtraction of member cost-share amounts.The incidence rate was 0.91 cases of SS per 1,000 enrollees, increasing with age. The mean age of SS patients was 50 years, and 53% were male. Approximately 63% received surgical intervention. Mortality was 21% during the first hospitalization and 36.1% at 1 year. During follow-up, 47.1% of survivors were rehospitalized. Mean index hospitalization length of stay and costs were 16 days and $26,820, with 1-year inpatient and outpatient costs totaling $48,996. Mean outpatient costs per survivor were $8,363, and mean per-patient-per month (PPPM) outpatient costs were $906. Total follow-up costs including rehospitalization were similar for nonsurvivors compared with survivors , but PPPM costs were higher for nonsurvivors .Incidence, hospital, and 1-year mortality rates were lower in this population compared with literature reports and were associated with a lower average age in this managed care population. Mean SS hospitalization costs were high, and nearly one half of survivors required rehospitalization within 1 year. Study results suggest the need to evaluate SS interventions for improvement in health outcomes and cost outcomes, particularly in postsurgical patients.


Flaviviridae, Retroviridae, and Poxviridae, the AUC of the RF model was greater than 0.8. Finally, the contribution of virus receptors to cross-species transmission was evaluated, and the virus receptor was found to have a minor effect in predicting the cross-species transmission of mammalian viruses. The study deepens our understanding of the mechanism of virus cross-species transmission and provides a framework for predicting the cross-species transmission of mammalian viruses.Most emerging viruses are spilled over from mammals. Understanding the mechanism of virus cross-species transmission and identifying zoonotic viruses before their emergence are critical for the prevention and control of newly emerging viruses. This study systematically investigated the host proteins associated with the cross-species transmission of mammalian viruses based on 1,271 pairs of virus-mammal interactions including 382 viruses from 33 viral families and 73 mammal species from 11 orders. Numerous host proteins were found to contribute to the cross-species transmission of mammalian viruses. Host proteins potentially contributing to virus cross-species transmission are specific to viral families, and few overlaps of such host proteins are observed in different viral families. Based on these host proteins, the random-forest (RF) models were built to predict the cross-species transmission potential of mammalian viruses. Moderate performance was obtained when using all viruses together. However, when modeling by viral family, the performance of the RF models varied much among viral families. In 13 viral families such as Emerging viruses pose serious threats to humans. Understanding the mechanism of virus cross-species transmission and identifying zoonotic viruses before their emergence are critical for the prevention and control of emerging viruses. This study systematically identified host factors associated with cross-species transmission of mammalian viruses and further built machine-learning models for predicting cross-species transmission of the viruses based on host factors including virus receptors. The study not only deepens our understanding of the mechanism of virus cross-species transmission but also provides a framework for predicting the cross-species transmission of mammalian viruses based on host factors. During the last 20 years, several global epidemic or pandemic diseases have been caused by emerging and re-emerging viruses, such as SARS-CoV , H1N1 viMany factors influencing the cross-species transmission of mammalian viruses have been reported, which can be roughly classified into two categories. The first category comprises macro-level factors including ecological, social-economic, evolutionary, and biological factors that determine whether the virus can access the novel host . In a prThe second category comprises molecular-level factors, including viral and host factors, which determine whether the virus can infect the host cell. In terms of viral factors, the viral receptor-binding protein and other viral proteins determine whether the virus can infect the host cells , 14. In This study systematically investigated the host proteins associated with the cross-species transmission of mammalian viruses by viral family and further evaluated the effect of host proteins and viral receptor proteins in predicting the cross-species transmission of mammalian viruses. It deepens our understanding of the mechanism of virus cross-species transmission and provides a framework for predicting the cross-species transmission of mammalian viruses.The workflow of the study is shown in The interactions between viruses and mammal species were derived from three sources: the Olival’s study, which curated 2,805 pairs of virus-mammal interactions from the literature , the VirThe proteome of mammal species, including humans, was obtained from the NCBI RefSeq database on 26 March 2021.Proteins in 73 mammal species were clustered by OrthoFinder (version 2.0)  with defhttps://pingouin-stats.org/index.html#)  was used to remove the influence of the phylogeny of mammal species on the correlations between viral cross-species transmission and protein groups, with cytochrome b as the covariable. The PSCC was calculated with the tool pingouin  . The P-venrichGO and enrichKEGG in the packages “clusterProfiler” (version 4.8.1) (The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of human proteins were conducted with the functions of n 4.8.1)  and org.n 4.8.1)  in R (ven 4.8.1) .BalanceRandomForest in the package “imbalanced-learn” (version 0.8.0)   in PythoThe leave-one-out test by mammal species was used to evaluate the performance of the RF model. In this approach, one mammal species was excluded from the data set each time, and the samples associated with the excluded mammal species were used as the testing data, while the remaining data were used as the training data. This process was repeated for each mammal species in the data set. The predictive performance of the RF model was measured by the area under the receiver operating characteristics curve (AUC).To reduce the bias of virus-host interactions in the modeling, samples from three mammal species in each order were randomly selected for modeling, and this process was repeated 1,000 times.k-Mi-Mj, the protein groups containing the receptors of Vk were identified; then, the pairwise sequence similarities between proteins in Mi and those in Mj were calculated for each protein group, the average similarity was computed for each protein group, and the maximum average similarity was considered the similarity between virus receptors in Mi and Mj (SMi-Mj); finally, SMi-Mj was used to predict whether the virus Vk had cross-species transmission between Mi and Mj.A total of 103 virus receptor proteins of 72 mammalian viruses were obtained from the ViralReceptor database . These rFlaviviridae, Picornaviridae, and Sedoreoviridae had higher number of interactions with mammals. For example, the Saint Louis encephalitis virus in the Flaviviridae family and the encephalomyocarditis virus in the Picornaviridae family infected 15 and 12 mammal species, respectively (Table S1). Regarding mammals, humans were the most infected by viruses (272 viruses), followed by the orders of Artiodactyla, Primates (excluding humans), and Rodentia. For example, the Bos taurus in the Artiodactyla was infected by 94 viruses from 20 viral families (Table S1).A total of 1,271 pairs of virus-mammal interactions were obtained, which included 382 viruses from 33 viral families and 73 mammal species from 11 orders . The correlations between protein groups and virus cross-species transmission were analyzed . When cooviridae .Coronaviridae had an average of two protein groups shared with other viral families. However, some viral families were observed to share a large ratio of protein groups with one or several other viral families. For example, the Nairoviridae shared 48 and 33 protein groups with Phenuiviridae and Sedoreoviridae, respectively  were only observed in one viral family. However, there were 21 protein groups observed in five or more viral families (Table S3). For example, the protein group with the human protein Transmembrane protein 250, which was present in most (eight) viral families, has been reported to promote virus proliferation.Herpesviridae, Filoviridae, Arenaviridae, Hantaviridae, Nairoviridae, Peribunyaviridae, Phenuiviridae, and Retroviridae . When considering all viruses together, the RF model performed moderately with a median AUC of 0.772 . When prHepadnaviridae and Kolmioviridae, the RF models had AUCs greater than 0.90, signifying the critical role of the virus receptor in virus cross-species transmission within these viral families.Virus receptors have been recognized as crucial factors in virus cross-species transmission. It is hypothesized that the more similar the virus receptors of two species, the more likely the virus crosses species between the two species. Accordingly, the protein sequence similarity between virus receptors of different mammals was used to predict the potential for mammalian virus cross-species transmission . When considering all viruses together, the RF model based on the virus receptor similarity only achieved an AUC of 0.588 . When anNewly emerging viruses pose serious threats to public health and have become a global concern in recent years. To effectively prevent and control newly emerging viruses, it is crucial to identify factors associated with virus cross-species transmission and develop computational methods for predicting virus cross-species transmission. Previous studies have reported several host factors associated with virus cross-species transmission –19. InThe study revealed that the host proteins associated with cross-species transmission of mammalian viruses were specific to viral families, with only a few overlapping proteins identified in different viral families. This finding indicates that the interactions between viruses and hosts are likely to be specific to viral families. Previous studies showed that the protein-protein interactions between viruses and hosts are virus-specific and even strain-specific , 34, as The virus receptor has been known to play a significant role in determining the susceptibility of hosts to viral infections. A previous study conducted by Cho et al. investigated the prediction of viral cross-species transmission infection propensities based on receptor similarity using 18 virus receptors . They foUnderstanding the interaction network between viruses and hosts may help much in clarifying the mechanism of virus cross-species transmission and improving the prediction model of predicting virus cross-species transmission , 36. DesPrimates and Artiodactyla because they have been more extensively investigated. Additionally, the absence of viruses in a species may be due to sampling bias, as it is challenging to determine definitively whether a virus did not infect a particular species. Further data and research are required to validate and refine the findings. Second, the models of predicting the cross-species transmission of mammalian viruses showed poor performances in some viral families, indicating that some factors contributing to the virus cross-species transmission may have been overlooked in these models. Integrating both macro-level and molecular-level factors could potentially enhance the predictive models for cross-species transmission of mammalian viruses.There were several limitations to this study. First, the interactions between mammals and viruses may be incomplete and biased. Most interactions are biased toward the Overall, this is the first study to systematically investigate the host proteins associated with cross-species transmission of mammalian viruses and predict the cross-species transmission of the mammalian virus based on both virus receptors and other host proteins. It deepens our understanding of the mechanism of virus cross-species transmission and provides a framework for predicting the cross-species transmission of mammalian viruses.


Geminiviridae encode replication-associated protein (Rep), which is a multifunctional protein involved in virus DNA replication, transcription of virus genes, and suppression of host defense responses. Geminivirus genomes are replicated through the interaction between virus Rep and several host proteins. The Rep also interacts with itself and the virus replication enhancer protein (REn), which is another essential component of the geminivirus replicase complex that interacts with host DNA polymerases α and δ. Recent studies revealed the structural and functional complexities of geminivirus Rep, which is believed to have evolved from plasmids containing a signature domain (HUH) for single-stranded DNA binding with nuclease activity. The Rep coding sequence encompasses the entire coding sequence for AC4, which is intricately embedded within it, and performs several overlapping functions like Rep, supporting virus infection. This review investigated the structural and functional diversity of the geminivirus Rep.Circular single-stranded DNA viruses of the family  Cressdnaviricota). Certain CRESS DNA viruses of eukaryotic origin encode only Rep and coat protein genes. Due to the HUH (two histidine residues separated by hydrophobic residues) endonuclease domain in the Rep, most DNA viruses replicate using a rolling circle mechanism (Geminiviridae replicate via rolling circle replication (RCR). In addition, the geminivirus Rep  interacts with the host DNA replication factors for virus DNA replication. The origin of the geminivirus Rep coding sequence is predicted from phytoplasma plasmids using the metagenomic sequence analysis (Single-stranded DNA (ssDNA) viruses infect a wide variety of living organisms. According to a recent metagenomic sequencing analysis, these intracellular obligate parasites are widespread and have a diverse host range . Followiechanism . Like otanalysis . HoweverGenome duplication during the cell cycle is highly regulated in all eukaryotes and is fundamental to preserving genome integrity . This duBecurto-, Begomo-, Capula-, Citloda-, Curto-, Eragro-, Grablo-, Maldo-, Mastre-, Mulcrile-, Ogun-, Topile-, Topocu-, and Turncurto-virus, based on the genome organization, host range, insect vector type, and phylogeny , or bipartite, with two ∼2.6 kb genomic components, DNA-A and DNA-B, together making up the genomic size of around ∼5.2 kb. DNA-A and DNA-B components are packed into twinned quasi-icosahedral virions  as one of the most numerous and diverse groups of plant viruses, causing a wide range of diseases in economically important plants . Naturalhylogeny . Members virions . Members virions . Geminiv virions . In addi virions . The DNA virions . Both DNAlphasatellitidae and Tolecusatellitidae, respectively (Nanoviridae and encode a nanovirus-like Rep (280–315 amino acids), which enables them to self-replicate. However, they require helper virus components for inter- and intracellular movement and, in exchange, provide helper viruses with selective advantages by acting as suppressors of gene silencing (Begomoviruses in the Old World (OW) and a few mastreviruses have been associated with ∼1.4 kb-sized ssDNA molecules referred to as alphasatellites and betasatellites  and receectively . Alphasailencing . Alternailencing .Tomato golden mosaic virus (TGMV) Rep overexpression in Nicotiana benthamiana plants supported efficient virus replication mutated for its Rep (Bean golden mosaic virus (BGMV) Rep resulted in partial resistance in bean plants to the respective viruses , which is a spliced version of C1 and C2 open reading frames (ORFs)  with sizes varying from 260 to 300 amino acids, transcribed from ORF C1  . In addim ORF C1 .The N-terminal of geminivirus Rep comprises three domains: DNA binding , DNA cleavage and ligation , and oligomerization  domains. It also contains the binding site for various host proteins, such as proliferating cell nuclear antigen (PCNA), retinoblastoma-related (RBR) protein, SUMO -conjugating enzymes (SCE), Geminivirus Rep-interacting kinase (GRIK), and adenosine triphosphatase (ATPase), as well as geminivirus protein REn. The DNA-binding and cleavage/ligation domains contain three motifs , which are involved in DNA binding, metal binding, and catalyzing endonuclease activity, respectively . FurtherBecurtovirus) and has been demonstrated to be indispensable for geminivirus genome replication as a mutation of the RXL motif failed to replicate the virus genome in N. benthamiana and fission yeast  Rep HUH endonuclease domain and the NMR structure of the catalytic domain of Tomato yellow leaf curl virus (TYLCV) Rep have been solved  in the N-terminal  Rep predicted the presence of a nuclear localization signal between the amino acid residues 244 and 273  and binding sites for RBR and GRAB (geminivirus RepA binding) transcription factors (Maize streak virus (MSV) RepA have shown that it is necessary for efficient virus DNA replication and is involved in ssDNA production  C4 was found to interact with and sequester the phosphorylation ability of glycogen synthase kinase 3 (GSK3) in N. benthamiana, a SHAGGY-like kinase and an inhibitor of cyclin CycD1;1, designated as SKη. This interaction leads to the accumulation of CycD1;1, which then forms a complex with CDK and induces cell division (S phase) that enables virus DNA replication (Beet severe curly top virus (BSCTV)-encoded C4-mediated upregulation of RING finger E3 ligase (RKP) in Arabidopsis plants is yet another strategy to induce cell cycle progression by Curtovirus , and CDK inhibitors (CKIs). Recently, lication . Beet sertovirus . Furtherrtovirus .Mungbean yellow mosaic virus (MYMV) targets BARLEY ANY MERISTEM 1 (BAM1), a receptor-like kinase that is a positive regulator of cell-to-cell movement of RNAi signals (Cotton leaf curl Multan virus (CLCuMuV) targets S-adenosyl methionine synthetase activity, a critical enzyme of the host methyl cycle, thus suppressing both transcriptional and post-transcriptional gene silencing (Tomato leaf curl New Delhi virus (ToLCNDV) C4 protein interacts with the host AGO2 protein and influences RNA silencing and cytosine methylation of the virus genome  AC4 has also been described as a suppressor of gene silencing  Rep-binding regions consist of direct repeat “GGTGTCT” elements in CR, as demonstrated by footprinting analysis (Mungbean yellow mosaic Indian virus (MYMIV) contains an additional iteron sequence downstream of CR  Rep self-interaction to form oligomers and their accumulation in the nucleoplasm  have shown that REn enhances the ATPase activity of Rep  and delta (δ) to the replicase complex and also interacts with virus Rep . In vitry of Rep . REn alsy of Rep . The abiy of Rep . Geminivy of Rep .Tomato yellow spot virus (ToYSV) DNA-B, which do not have identical Rep-binding regions, demonstrates that trans-replication of only its cognate DNA-B molecule is not a strict rule  and Ageratum yellow vein betasatellite (AYVB) can be trans-replicated using CLCuMuV, Ageratum yellow vein virus (AYVV), and Eupatorium yellow vein virus (EpYVV), but not by Honeysuckle yellow vein virus (HYVV) (Tobacco curly shoot betasatellite (TbCSB) and Tomato yellow leaf curl China betasatellite (TYLCNB) can be trans-replicated by their non-cognate helper viruses, but when co-inoculated, cognate betasatellites predominated over non-cognate partners in the later stages of infection  . It has nfection . These sTobacco curly shoot virus (TbCSV)  . Competi (TbCSV) . SequencArabidopsis plants mutated for methylation such as cytosine methyltransferases (CMT) and histone methyltransferases (MET) supported high levels of geminivirus DNA replication, and virus DNA isolated from such plants was less methylated , Gossypium mustelinium symptomless alphasatellite (GMusSLA), and Cotton leaf curl Multan alphasatellites (CLCuMuA), are known PTGS suppressors . HoweverTomato yellow leaf curl Sardinia virus (TYLCSV) responsible for its transcription autoregulation activity  interact with tomato, tobacco, and pea PCNA, respectively (Pedilanthus leaf curl virus (PeLCV) and CLCuMuV, has been shown to interact with not only plant PCNA but also yeast PCNA, suggesting a high conservation of Rep binding domains in PCNA among eukaryotic species , a single-stranded (ss) DNA-binding protein primarily involved in replication, repair, and recombination  to the 3′ end of nicked geminivirus DNA, assisting in the assembly of the virus DNA replication elongation complex in wheat cells . In vitrbination . The 32 lication . Further unclear .Cabbage leaf curl virus (CaLCuV) and TGMV Rep interact with the histone-3 (H3) protein of Arabidopsis. This interaction might help displace histones from the virus nucleosomes, thus helping in virus DNA replication and transcription processes (N. benthamiana-encoded histone ubiquitination-1 (NbHUB1) and ubiquitin-conjugating enzyme 2 (NbUBC2), and recruits these proteins on the promoter sites at virus minichromosomes   that attrad51 and rad54 displayed reduced virus DNA replication while it was restored upon expression of RAD51 and RAD54 proteins. The binding domains have been mapped to the N-terminal of RAD54 and the oligomerization domain of MYMIV Rep  increases Euphorbia yellow mosaic virus (EYMV) DNA replication  in addition to RCR . Rep intYMIV Rep . In vitrlication , 2016. Tlication .African cassava mosaic virus (ACMV) interact with sumo-conjugating enzyme 1 (SCE1) to enhance virus DNA replication  funded by the Ministry of Oceans and Fisheries, Korea (20210466).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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Viruses are widespread in the environment, and many of them are major pathogens of serious plant, animal, and human diseases. The risk of pathogenicity, together with the capacity for constant mutation, emphasizes the need for measures to rapidly detect viruses. The need for highly sensitive bioanalytical methods to diagnose and monitor socially significant viral diseases has increased in the past few years. This is due, on the one hand, to the increased incidence of viral diseases in general , and, on the other hand, to the need to overcome the limitations of modern biomedical diagnostic methods. Phage display technology antibodies as nano-bio-engineered macromolecules can be used for sensor-based virus detection. This review analyzes the commonly used virus detection methods and approaches and shows the prospects for the use of antibodies prepared by phage display technology as sensing elements for sensor-based virus detection. Sulfolobus islandicus rod-shaped virus 2 can be used in bioconjugation chemistry ..26].−1 [An optical immunosensor was developed to detect the NS1 antigen  as a biomarker of dengue virus in clinical samples obtained early in infection. The principle of operation is based on the detection of the NS1 antigen by immunofluorescence by using fluorescein-5-isothiocyanate (FITC) conjugated to an IgG antibody. The probe was highly reproducible  and sufficiently stable for 21 days at 4 °C, with a detection limit of 15 ng/mL−1 .Guliy et al.  reportedOwing to their excellent chemical stability, acoustic biosensors are most frequently made with piezoelectric crystals such as quartz, lithium niobate, and lithium tantalate. An attractive approach to creating a family of sensors characterized by high sensitivity, rapidity of analysis, low cost, and tiny sizes is the excitation of acoustic waves in a piezoelectric material. Some techniques rely on membranes or receptor antibodies placed on a piezoelectric waveguide or the resonator surface . For insFor example, bacteriophage M13 was identified in real-time by Tamarin et al. , who useAn electroacoustic sensor platform was used for the immunodetection of the transmissible gastroenteritis virus of swine (TGEV) . RecentlSignificantly, all immunochemical methods, including biosensor-based ones, depend primarily on the quality of the specific antibodies used to make diagnostic systems. Antibody selection targets range from peptides and recombinant proteins to viral or virus-like particles . Virus-sHistorically, the first source of antibodies was sera from immune animals and humans. The antibodies obtained contain a set of immunoglobulins of different classes and subclasses that specifically recognize their antigen. Different serum antibodies recognize several sites (epitopes) of the antigen. Antibody preparation was then aided by hybridoma technology , which m-Antibodies can be selected in vitro, and animal immunization can be omitted;-No need to use laboratory animals or maintain long-term cultures of eukaryotic cells;-The preparation of individual clone producers of miniantibodies takes 10–14 days, against the several months that hybridoma technology takes;-Antibodies are relatively easy to prepare, and their cost is low;-It is possible to make hybrid molecules with marker proteins  in a short time;-It is possible to prepare antibodies to autoantigens, weakly immunogenic compounds, and toxins;-The method can be used in immunotherapy.To resolve these problems, methods for the molecular cloning of antibody gene fragments have been put forward. One such method is antibody phage display, proposed by . The antTemperate filamentous phages  are most commonly used in phage displays. Their virion contains circular single-chain DNA, whose genetic organization and sequence are known by many members of this phage group. The genome size is about 6000 nucleotides. The five structural proteins that make up the capsid of a viral particle are organized similarly in all members of the group .-The immobilized antigen is incubated with library clones;-Particles of the phage that have not yet attached to the antigen are washed away;-The bound phage particles are eluted;-E. coli);The selected clones are infected with bacterial cells (-The affinity clones are amplified and isolated.Human phage display libraries or libraries from other species are used to make antibodies. These libraries will be utilized for the in vitro selection (biopanning) of the target molecule. The following actions are part of the process of choosing phage library components:The immunogenicity of the antigens, how they are immobilized, and the quantity of biopanning rounds all affect how long it takes to create an antibody. That is why the process can take a few hours or several days. After biopanning, the monoclonal phage antibodies are analyzed by various immunochemical assays, in particular, ELISA. Subsequently, the genes of the selected antibody fragments can be isolated from the phage particles for subsequent cloning and expression. -The genes of scFv, Fab, or other fragments are cloned from immune or undamaged humans, mice, rabbits, chickens, pigs, dogs, monkeys, sheep, or cows’ B lymphocyte mRNA;-These genes, along with the gene coding for the capsid protein , are introduced into the phagemid in a single translation frame;-E. coli cells by using the generated phagemid repertoire.Phagemid genes are expressed, and virions are put together in infected The following is the general process for creating a combinatorial phage library:Foreign antibody fragments will be exposed as part of the capsid proteins of the virions. Depending on the chosen vector system, this step takes place with or without the use of helper phages.-Owing to the Fc region of the intact antibody, the tiny size of the antibody fragments is typically accompanied by reduced nonspecific binding;-The biosensor can immobilize phage antibodies more densely;-E. coli cells, which significantly lowers production costs because no specialized equipment is needed for the culture of hybridoma cells [Phage antibodies, as opposed to full-length antibodies, are produced by ma cells .In this way, a population of bacteriophages is prepared, each exhibiting a specific antigen-binding domain on its surface ,74. The Phage display technology antibodies as nano-bio-engineered macromolecules can be used for sensor-based virus detection. In order to recognize lethal viruses such as Zika, Ebola, Hendra, Nipah, Hanta, Middle East respiratory disease (MERS), and SARS, phage display has been utilized to create new diagnostic tools ,77. In tBy now, a huge pool of antibodies against various viruses has been obtained by phage display, by using naïve or immune libraries. From naïve antibody gene libraries, antibodies against human pathogenic viruses such as SARS coronavirus, dengue virus, influenza virus, Venezuelan equine encephalitis virus, norovirus, and hepatitis C virus have been developed by using recombinant viral proteins or complete viral particles ,79. OtheRotavirus gp6, H5 hemagglutinin to inhibit H5N1 influenza virus replication, and VHH that recognizes the tail of infectious phage in Lactococcus bacteria.By using phage display, potential therapeutic agents are being developed for other human coronaviruses, such as SARS  and MERSExamples of the use of phage antibodies for virus detection using classical methods are presented in Undoubtedly, phage antibodies are an excellent alternative to classical antibodies as sensing elements in virus diagnostics by biosensors . For exaA sandwiched phage-based enzyme-linked chemiluminescence immunoassay was created by Liu et al.  by usingAfter three rounds of biopanning, Yang et al.’s  screeninA human combinatorial scFv antibody library was used to retrieve a panel of recombinant single-chain antibodies (scFvs) against structural proteins of tomato spotted wilt virus via phage display . Using pTo distinguish avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2) (whose nucleoproteins are 90–94% similar in amino acid sequence), Yu et al.  developeThe HBsAg of the hepatitis B virus was demonstrated by ELISA to have a high affinity for the generated phage scFvs, which allowed them to bind to the antigen on the membrane of the infected cells. One of the clones was capable of being internalized into HepG2.2.15 cells that were HBsAg positive, according to indirect fluorescent labeling examination. The ability to internalize phage antibodies to deliver medications to cells that are infected with the hepatitis B virus may be highly promising .By using phage display, methods have been developed to generate specific single-chain variable fragments (scFvs) against dengue fever virus  and infePhage antibody-based ELISA versions have been created for the detection of human disease-causing viruses such as HIV, herpes simplex virus, poliovirus, polyomavirus, rabies virus, respiratory syncytial virus, rotavirus, Sin Nombre virus, Usutu virus, West Nile virus, yellow fever virus, Zika virus, human metapneumovirus, Japanese encephalitis virus, MERS, norovirus, paramyxovirus, chikungunya virus, Ebola virus, enterovirus 71, Epstein-Barr virus, hantavirus, human cytomegalovirus, Hendra virus, Nipah virus, and hepatitis A, C, and E viruses .A useful natural resource for investigating polyvalent interactions with particular antibodies is bacterial viruses. Such research is very useful in the real world and has been used to create improved virus detection methods. For example, Guliy et al.  showed tThe possibility of using phage display antibodies in veterinary medicine was demonstrated in . The greOne of the most destructive viral illnesses in the dairy and meat sectors is foot-and-mouth disease (FMD). It is known that there are seven FMD serotypes: O, A, C, Asia1, SAT1, SAT2, and SAT3. Therefore, a quick and precise way of differentiating between vaccinated and infected animals is essential to the effectiveness of diagnosis and vaccination. Recombinant chicken scFvs against the FMD 3ABC protein have been produced and demonstrated to be effective for separating infected from vaccine-exposed animals via phage display ,207. AddThe promise of phage antibodies as an experimental tool has been demonstrated to visualize  and diagPossibilities have been shown to detect plant viruses  and zoonotic viruses  with phage antibodies and ELISA ,74.Norovirus, Maedi-visna, SARS-CoV, Sin Nombre, and duck hepatitis A viruses have been identified with the help of dot-blot immunoassay and phage antibodies ,74. PhagNote that surface plasmon resonance (SPR) sensors for the detection of chemical and biological species are often the “gold standard” ,211,212.As can be observed from the data displayed, biosensors enable a large decrease in the analysis time owing to the relative simplicity of the procedures, are quite sensitive, and require little pretreatment of the material under examination. The instrumental implementation of these methods should ensure high accuracy of measurements, and in turn, measurements should be made automatically by moderately skilled personnel. When biosensors are designed for the detection of viruses and virus-like particles, the following criteria should be taken into account: property of being all-purpose, ability to be used with various objects, rapidity of detection, specificity, sensitivity, portability, simplicity, ability to use small sample volumes, and low cost .The selection of antibody fragments from phage display libraries thus offers the potential to access very large numbers of molecules with different binding specificities quickly and cheaply, avoiding the need for animal immunizations. The scFv can be genetically engineered to produce tailored constructs as described above. Such phage antibodies can be coated directly onto metal, plastic, silica or carbon surfaces by simple adsorption, which, as discussed above, is adequate for biosensor-based assays.Viruses are pervasive life forms that need a host to reproduce. From bacteria to plants and animals to viruses, a wide variety of organisms may become infected. The genome, which is represented by double- or single-stranded DNA or RNA, and the capsid are the two primary structural parts of viruses. A small number of proteins encoded by the viral genome are present in numerous copies to create the extremely symmetrical capsid . MethodsPhage display is a powerful tool for target ligand selection owing to its being simple, highly effective, rapid, and cheap. Efficient biopanning selection leads to the isolation of ligands with unique, specific, and desired functional characteristics. Phage antibody display offers multiple platforms for the use of antigen-binding; however, the diversity and stability of the library still need to be improved.In the past few years, the need for highly sensitive bioanalytical methods to diagnose and monitor socially significant diseases has increased. This is due, on the one hand, to the growth of the disease in general, including the unprecedented spread of the new coronavirus infection, SARS-CoV-2, and, on the other hand, to the need to overcome the limitations of current biomedical diagnostic methods. For example, because of puncture invasiveness, a tissue biopsy cannot always be performed, and the results of a single biopsy often cannot provide sufficient information in real-time to diagnose the disease. PCR is currently one of the most popular tools for the rapid detection of viral infections. Nucleic acid-based virus detection usually provides high sensitivity but may require trained personnel and be time-consuming and expensive. The use of isothermal amplification systems may reduce equipment costs, making these systems indispensable when high-performance and rapid cycling testing are required. Alternatives to PCR are immunoassays, which offer reliability and cost-effectiveness. In addition, some immunoassays can be modified with lateral flow technology, which greatly speeds up the generation of results. However, immunoassays are usually inferior in sensitivity to PCR. Along with these methods, the use of next-generation sequencing can provide promising results. In addition, the ability to sequence a large number of viral genomes will provide researchers with expanded information about them and will help in tracing infections.Characteristics such as high throughput, ease of use, and short running time play an important part, especially in viral outbreaks, in addition to the general requirement for the accuracy, validity, and specificity of viral assays. This strategy enables simple, cheap, rapid, and sensitive detection of specific pathogens, which shows great potential in virus analysis in situ.The growing advances in phage display and the use of antibodies in the diagnosis of viral diseases suggest this technology will be improved further. This progress will gradually drive innovation to understand the mechanisms involved in infection and to design post-exposure therapies for SARS-CoV-2 and other viral infections.