Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) has emerged as a key upstream regulator of cell death and inflammation. RIPK1-mediated signaling governs the outcome of signaling pathways initiated by tumor necrosis factor receptor 1 (TNFR1), Toll-like receptor 3 (TLR3), TLR4, retinoic acid-inducible gene 1 (RIG-I)/melanoma differentiation-associated protein 5 (MDA-5), and Z-binding protein 1 (ZBP1) by signaling for NF-κB activation, mitogen-associated protein kinase (MAPK) and interferon regulatory factor 3/7 (IRF3/7) phosphorylation, and cell death via apoptosis and necroptosis. Both cell death and inflammatory responses play a major role in controlling virus infections. Therefore, viruses have evolved multifaceted mechanisms to exploit host immune responses by targeting RIPK1. This review focuses on the current understanding of RIPK1-mediated inflammatory and cell death pathways and multiple mechanisms by which viruses manipulate these pathways by targeting RIPK1. We also discuss gaps in our knowledge regarding RIPK1-mediated signaling pathways and highlight potential avenues for future research. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) was first discovered as a protein that interacts with the death receptor, Fas , 3. SincRIPK1 is a member of the receptor-interacting serine/threonine family. RIPK1 harbors an N-terminal kinase domain, a RIP homotypic interaction motif (RHIM)-containing intermediate domain (ID), and a C-terminal death domain (DD) , 5 Fig.. The kinTumor necrosis factor alpha (TNF-α) ligation with TNF receptor 1 (TNFR1) initiates signaling cascades leading to NF-κB activation, MAPK activation, extrinsic apoptosis, and necroptosis . RIPK1 a9–The role of RIPK1 in TNF-α signaling is well studied. Upon TNF-α ligation, TNFR1-associated death domain protein (TRADD) binds to the cytoplasmic death domain (DD) of TNF receptor 1 (TNFR1). TRADD recruits TNF-associated factors (TRAF2/TRAF5) along with RIPK1 and cIAP (cellular inhibitor of apoptosis) proteins. Ubiquitination of RIPK1 by TRAF2 and/or cIAPs creates a platform for recruiting transforming growth factor β-activated kinase 1 (TAK1), TAK binding proteins (TAB1 and TAB2), and the IκB kinase (IKK) complex  –18. This–MAPKs (mitogen-associated protein kinases) consist of three groups, Erk1/2 , JNKs , and p38 , 22. Therip1 gene (which codes for RIPK1) in mouse embryonic fibroblasts prevented p38 MAPK activation and IL-6 production in response to TNF-α  can lead to potent innate immune responses via NF-κB activation. TLR4, the sensor for bacterial lipopolysaccharide (LPS), triggers NF-κB activation via either MyD88 (myeloid differentiation primary response gene 88)-dependent or TRIF (TIR domain-containing adapter inducing interferon β)-dependent signaling pathways , 30. TLRRIPK3 has been shown to inhibit RIPK1-mediated NF-κB activation by competitive binding to TRIF downstream of TLR3 and TLR4 . This RITLR3 and TLR4 activate signaling pathways through IRF3/IRF7 phosphorylation that culminate in the production of IFNs and IFN-inducible genes, which are central to antiviral innate immune responses . Even thThe RNA helicases RIG-I (retinoic acid-inducible gene 1) and MDA-5 (melanoma differentiation-associated protein 5) are known to initiate signaling pathways culminating in the production of type I interferons  and proinflammatory cytokines during the early stages of viral infections . RIG-I m42–RIPK1 is involved in NF-κB, MAPK, IRF3, and IRF7 activation downstream of RIG-I and MDA-5. RIG-I and MDA-5 signaling merge at the mitochondria through MAVS , which is located at the mitochondrial membrane . TNFR1-aZBP1 (Z-binding protein 1), also known as DAI (DNA-dependent activator of IFN regulatory factors) or DLM1, was initially recognized as a sensor for double-stranded DNA (dsDNA) but later found its role as an RNA sensor during virus infections –58. Furt–−/−RIPK1 and −/−ZBP1 cells relative to wild-type (WT) or KD/KDRIPK1 bone marrow-derived macrophages (BMDM) infected with IAV  exhibited lower levels of Irg1 mRNA and higher ZIKV titers in brain homogenates and mortality relative to wild-type controls  replication in neurons by altering cellular metabolism. Mice expressing a kinase dead version of RIPK1  M45.Viruses are obligate intracellular pathogens. Hence, cell death can be an important immune defense against infection. RIPK1 regulates cell death pathways known as “ripoptocide” involving both apoptosis and necroptosis . ApoptosNecroptosis is considered an antiviral defense mechanism because it can initiate a strong proinflammatory response. Necroptosis is characterized morphologically by swelling of organelles followed by plasma membrane rupture –73. Necr–Furthermore, ZBP1 is also known to trigger virus-induced necroptosis by activating RIPK3 , 78. RIPEven though RIPK1 participates in extrinsic apoptosis and necroptosis, the loss of RIPK1 also sensitizes cells to TNF-α-induced apoptosis , 82, perRIPK1 is placed at a crucial point controlling cell death and inflammation, governing the outcome of signaling pathways initiated by TNFR1, TLR3, TLR4, RIG-I/MDA-5, and ZBP1 and leading to NF-κB activation, MAPK, and IRF3/7 phosphorylation. Activation of these pathways results in release of proinflammatory cytokines and interferons, setting an antiviral state, and in destruction of the replication niche of obligate intracellular pathogens via cell death. Therefore, RIPK1 is as an attractive target for inhibition by different virus families.Herpesviridae family have double-stranded DNA genomes. They are categorized into alpha-, beta-, and gammaherpesviruses based on their host range, genome, and replication strategies , and Zika virus (ZIKV). Tick-borne viruses include tick-borne encephalitis virus, Powassan virus, louping ill virus, and Omsk hemorrhagic fever virus . WNV is RIPK1 together with RIPK3 signals for chemokine expression and for recruitment of infiltrating antiviral leukocytes in the central nervous system during WNV infection. Mice expressing a kinase dead form of RIPK1 exhibited increased susceptibility to WNV infection . Even thKD/KD) had higher viral titers in whole-brain homogenates upon infection with ZIKV. Inhibition of kinase activity in vivo by treatment of wild-type mice with pharmacological inhibitors also increased brain viral burden. Furthermore, RIPK1KD/KD neuronal cultures exhibited enhanced replication of ZIKV relative to wild-type controls. Interestingly, no differences were observed in ZIKV replication in primary BMDM or dendritic cells from wild-type versus RIPK1KD/KD mice or with inhibition of the kinase activity of RIPK1, suggesting that these effects are cell-type specific  plays an important role in the virus life cycle by cleaving viral polypeptides Gag and Gag-Pol into mature proteins . One stuThe HTLV-1-encoded Tax protein is a multifunctional protein that is crucial for T cell transformation by HTLV-1 . Tax inhHRV is the major causative agent of the common cold and asthma exacerbations . There aFiloviridae family  , 132. AtIn this review, we have discussed various strategies exploited by viruses to inhibit RIPK1-mediated signaling. Viral modulation of RIPK1-mediated signaling may inhibit NF-κB responses, MAPK signaling, IFN induction, and cell death. It appears from the literature cited above that most viruses benefit by inhibiting these pathways. Understanding the impact of inhibition of RIPK1 signaling on the host and the virus may facilitate the development of new immunotherapeutic strategies against virus infections.


We performed measurements both at random locations as well as close to the virus entry by conjugating viruses to nanodiamond sensors. We observed alterations of T1, which represent the intracellular free radical concentration during the viral replication process. Moreover, relaxometry is also used to monitor real-time free radical variation during the early infectious process.Although viruses are known to modify the free radical concentration in infected cells, the exact location and concentrations of such changes remain unknown. Although this information is important to understand the virus pathogenesis and design better anti-viral drugs or vaccines, obtaining it with the conventional free radical/ROS detection techniques is impossible. Here, we elucidate the utility of diamond magnetometry for studying the free radical response of baby hamster kidney-21 cells upon Semliki Forest virus infection. Specifically, we optically probe the alterations in free radical concentration near infectious viruses via measuring the spin–lattice relaxation (T Redox imbalance in viral infections is an extensively studied topic. Most studies on oxidative stress in viral infections do not differentiate between paramagnetic and non-paramagnetic reactive molecules. Instead they assess the amount of the reactive molecules in cells or evaluate their overall molecular and enzymatic response to probe the infection induced redox imbalance. However, this approach is not always useful and sometimes type/nature of an individual reactive species needs to be considered. For instance, although the amount of oxygen radicals upon influenza virus infection has been shown to increase , similarConventionally, fluorescence-based assays ,11 or UVHere, we demonstrate that relaxometry can be an excellent technique to study intracellular free radicals upon viral infection. In free radical detection, this method offers distinct advantages in terms of biocompatibility, sensitivity , resolut1) of NV ensembles embedded in an intracellular FND conjugated to SFV as well as bare FNDs. Moreover, the location and duration of host cellular radical response during viral infection, and the impact of viral infection on the cytoplasmic transport is elucidated for the first time.Here, we optically probe the free radical response of host baby hamster kidney-21 (BHK-21) cells upon Semliki Forest virus (SFV) infection by measuring the relaxation time  to conduct the pulsing sequence shown in 2.1.2Using a bright field camera (Thorlabs) we selected FND particles  well inside a cell with a brightness >2 million photon counts/s. These nanodiamonds are very well characterized in the literature . To avoi2.1.31, we use the spin relaxometry protocol developed by Tetienne et al. [1 measurement we first polarize the NV centers ensemble in the 70 nm FND ingested by the BHK-21 cells using 5 μs pulse of a 532 nm laser. Then, we turn the laser off for a predefined time interval, known as dark time, which varies between 0.2 μs to 106 μs. The laser pulse sequence used in this work for exciting the NV ensemble is shown in To experimentally determine the Te et al. . In shor2.1.41. The model we used to fit the T1 data was established earlier [Once the data acquisition is complete, the data was processed further to quantify the T earlier  and is d1 ((Tb) deeper in the crystal or less perturbed) [1 time was selected for quantification because it is more sensitive to changes of the NVs' surrounding. This model and some of the experimental parameters was determined by Perona Martinez et al. [Technically there are hundreds of NV centers in every particle. To simplify the fitting procedure the relaxation time of the ensemble is approximated to have two components: the NV centers with shorter  and one with longer Trturbed) . Betweenz et al.  by testi2.22. All the culture medium components were purchased from Gibco, Life Technologies, the Netherlands. We used active SFV -1640 medium  supplemented with 10% fetal bovine serum (FBS), 1% Penicillin/streptomycin and 1% Glutamax at 37 °C and 5% CO in Ref. ) and ina50 titer: 2.8 × 1010 TCID50/mL) was labeled with 3,3′-Dioctadecyloxacarbocyanine perchlorate (DiO)  following the protocol described by Hoornweg et al. [50) assay where the titer was found to be 2 × 108 TCID50/mL post labeling. Individual virus particles labeled with DiO were further characterized by fluorescence microscopy (Delta Vision Elite). For the microscopic analysis, 100 μl of 100-times diluted DiO-labeled SFV (DiO-SFV) solution were added to one quarter of the glass bottom Petri dish and imaged with a 488 nm laser. Emission was collected by a 100 × oil immersion objective with a numerical aperture of 1.40 and imaged through a FITC filter and a charge-coupled-device camera. Image analysis (intensity-particle number) was carried out by using Particles Analyzer plugin of ImageJ  as shown in To prepare the fluorescently-labeled virus, sucrose purified SFV  were added to 10 μL SA-FND , then the mixture was incubated at 25 °C for 2 h. The SFV-FND conjugates were used for further experiments without further purification. To examine the conjugating efficiency of SA-FND with biotinylated SFV, the SFV was firstly modified by biotin, then labeled by DiO. The resulting DiO labeled biotinylated SFV was then conjugated to SA-FND using the same protocol. After this, the mixture (DiO-SFV-FND) was diluted 10 times by PBS and examined by fluorescent microscope (DeltaVision Elite) through FITC and A594 filters, followed by image analysis using FIJI.The biotin-streptavidin technique was used to achieve SFV conjugation with FNDs because of its specificity and robustness . Specifi2.450 assay. TCID50 is the dilution of the virus stock required to infect 50% of the given cell population. In this assay, 40,000 cells plated in flat-bottom 96 well plates (Corning Costar) were infected with 10−1 through 10−12 dilution of SFV stock solution made with 2% (FBS) RPMI medium. At 1-h post infection (hpi), 150 μl of 10% (FBS) medium was added over the cells. Every 24 h until 72 hpi, the cells were examined for signs of SFV induced cytopathological effects (CPE) using a light microscope (Leica DMIL LED). The numbers of live and dead wells were counted and further used to calculate the TCID50 of the initial virus stock solution by the Spearman-Kärber algorithm given by Hierholzer and Killington [The virus titer of the SFV  stock solution was determined using the TCIDllington .2.5The general intracellular free radical response of BHK-21 cells was measured by sequentially incubating FND and SFV with host cells. One day before the experiment, 1 μg/ml 70 nm FND suspension (10% FBS + 90% RPMI-1640 medium) was added to 20,000 cells in one quarter of a glass bottom Petri dish . The FND suspension was made using a protocol established in our group ,20. ThenTo investigate the intracellular free radical response near the viral particles, virus-diamond conjugates (SFV-FND) were used to infect host cells. 40,000 BHK-21 plated in a four-quartered glass-bottom Petri dish  were incubated with SFV-FND with MOI = 20. In this experiment, SA-FND without viral particles treated BHK-21 cells was set as a control group. Here three experimental groups were cells treated with (i) infectious SFV-FND, (ii) inactivated SFV-FND and (iii) SA-FND at the 0 h time point.1 measurement. At every timepoint , we removed one Petri dish from the incubator and measured T1 relaxation on four different FNDs ingested by four different cells in the same Petri dish. Three measurements on a single Petri dish were completed within a maximum of 1 h after removing it from the incubator. Such an experiment was repeated three independent times per group.At 1 hpi, the infection medium was removed, and cells were supplemented with 400 μl of 10% medium. After this, cells were maintained in the incubator except during the treatment or the T2.61 measurement cycle for 85,000 times in 85 min using the same particle (as described in Section S10). For comparison, a rolling window experiment was also performed on the inactive SFV-FND conjugates or only SA-FND attached BHK-21 cells. Each experimental group was repeated 9 times.The real-time variation of intracellular free radicals near viral particles was investigated by two steps. First, seeded BHK-21 cells were incubated with 100 μL SFV-FND  for 45 min at 4 °C. Later, unattached SFV-FND were removed by washing the cells three times with cold RPMI. BHK-21 cells were kept on an ice bath with cold cell culture medium until further experiments. Then we applied the T1 in healthy and treated cells. Details of the algorithm are explained in www.graphpad.com).The data were analyzed using a “moving window algorithm” to determine the temporal evolution of T2.77 TCID50/ml SFV equiv.) was added to BHK-21 cells and cells were incubated for 45 min at 4 °C. Later, unattached virus or conjugates were removed by washing the cells three times with cold RPMI. Subsequently, the temperature was rapidly elevated to 37 °C using a thermostatic stage. This temperature was maintained throughout the experiment. The moment of the temperature shift to 37 °C is referred to as 0 min time point. DiO-SFV was detected with a FITC filter, while DiO-SFV-FND was detected with FITC and A594 filters, separately. An image series of the fluorescent emission was recorded with a charge-coupled-device camera at 1 frame per 5s for a total of 25 min. Before and after fluorescence imaging, the localization of the nucleus and plasma membrane of the cell was determined by differential interference contrast (DIC) imaging. As shown in To investigate how much time is required for SFV and SFV-FND to enter the cells, the virus-tracking experiments were performed. Briefly, BHK-21 cells were seeded one day before the experiment. Next day, cells were washed twice with cold phenol-red RPMI. Glucose oxidase (GLOX) was added to prevent photo toxicity as elaborated in the literature ,31. SubsAs a negative control, BHK-21 cells were treated with 20 mM ammonium chloride (Sigma) before incubation with virus or virus-diamond conjugates ,31. To m2.81 experiments on SFV-FND, BHK-21 cells were incubated with DiO-SFV-FND. After co-culturing for different times , the cells were fixed with 3.7% paraformaldehyde solution for 15 min, followed by staining the endosomes using EEA1 monoclonal antibody , and goat anti-Rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 350 , respectively. 0 hpi samples were obtained by attaching DiO-SFV-FND on BHK-21 cells on an ice bath for 45 min, immediately followed by the same fixing and staining protocol. The cells were then imaged using a fluorescent microscope (DeltaVision Elite). Signals from DiO, FND, and stained endosomes were obtained with FITC, A594, and DAPI filters. Images were analyzed using FIJI to determine the relative position for conjugates and endosomes.To obtain the position information of the conjugates, especially the location of FND where we carried out our T33.1Togaviridae family and one of the least pathogenic alphaviruses for humans [SFV is a positive-stranded RNA virus of the genus Alphavirus of the r humans . Its popr humans . Likewisr humans . Hence, Section S4.Next, we briefly explain the relevance of relaxometry in free radical sensing. A schematic representation of probing the general intracellular free radical response (with bare FNDs) and free radical response near viral particles (with FND-SFV conjugates) is shown in 3.21 experiment was done on an SFV-FND conjugates. The number of viral and diamond particles was calculated using the methods as described by Hilde et al. [The biotin-streptavidin interaction is widely used in biological experiments, especially in the biohybrid materials field ,34. Heree et al.  and Barte et al. . It can 3.39 TCID50/mL, we measured how long it takes for a virus particle to enter the BHK-21 cells. Here we used a method previously reported by Hoornweg et al. [4Cl, which inhibits the virus fusion [After determining the titer of the virus stock solution to be ∼2 × 10g et al.  In partis fusion . This is4Cl also inhibits the fusion of SFV-FND. In conclusion, the characteristic timing of early infection of the virus is similar for conjugates. There is around 4 times fluorescence increase for DiO-SFV's fusion, while there is only 2 times increase for the DiO-SFV-FND group , we continuously track the movement of the FND within the cell cytoplasm. Specifically, after every 3 to 5 s, we scan the fluorescence intensity within the 2 × 2 × 1 μm volume around the last known position of the particle. Subsequently, the new position of the FND is determined in two steps- (i) using a gaussian approximation to determine the brightest voxel in X and Y direction (ii) finding the brightest voxel at different Z coordinates for the X and Y coordinates obtained in the previous step. Tracking FND's movement within the cytoplasm allows us to further investigate the effect of viral infection on cytoplasmic transport. Diffusion of viral genetic material within the cytoplasm has been an important topic of experimental or computational investigation [While applying the pulsing sequences on an intracellular FND for measuring the relaxation time  would differ in T1. From the results it can be clearly seen that all the points form a dense cluster for the negative control group where T1 and the maximum displacement varies between 100 and 200 μs and 2–4 μm respectively. On the other hand, two components influencing the maximum displacement are (i) physical movement of an FND and (ii) an uncertainty in optical refocusing deployed for diamond tracking. We recorded a maximum displacement of ∼ 1–2 μm even for a stationary particle placed in a dry Petri dish. A similar cluster was observed for the inactivated virus and infectious virus group. Interestingly, the data points in those two groups were found to be more scattered compared to the negative control group indicating that FND movement in the cytoplasm is elevated upon introduction of the external stimulus. In 1 x displacement plane is not divided in “distinct colors” which means that no correlation between FND movement and the hpi can be drawn.Cytoplasmic transport can be assessed by a variety of ways such as quantifying the particle velocity, diffusion rate or displacement. Here, we quantify the maximum diamond displacement within the cell as shown in This method can easily be extended to both quantitatively and qualitatively investigate the cytoplasmic transport. Due to FND's photostable fluorescence, the same FND can be tracked continuously before and up to ∼24 h (based on the work in our group) after adding the virus. Uncertainty in optical refocusing can also be minimized by suitably modifying the tracking algorithm. Such measurements can potentially reveal the diffusion rate and particle velocities at high time resolution.3.51 experiments, BHK-21 cells were incubated with DiO-SFV-FND for different times, followed by a fixing and staining process. For the control group, the uptake of DiO-SFV-FND conjugates was blocked when the temperature was lowered to and kept at 4 °C for 1 h. As shown in In the previous section we investigated free radical generation at a random location which is not necessarily close to the point of virus entry. Here, virus-diamond conjugates were formed to investigate the intracellular free radical response near viral particles, as diamond can detect the magnetic noise within 10 nm range. To clarify the position of SFV-FND conjugates and the location where we carried out our T3.61 values detected using SFV-FND are more affected by the infectious events. In 1 value at 2 hpi (145 μs), compared with 0 hpi (220 μs). At 2 hpi is the time when SFV is completely uncoating. The latent period during the growth of SFV was 3 h. However, the traditional method based on RNA or protein techniques cannot detect the potential crisis, as viral replication and other activities can evade detection of immune responses from host cells, while relaxometry is sensitive to relatively early processes of radical formation. 1 values  at 6 hpi (91 μs). This is also the timing when the viral load is greatest as described by Kaariainen et al. [We investigated the intracellular free radical response near viral particles using SFV-FND conjugates at discrete time points post SFV infection with MOI 20. Comparing with n et al. . Accordin et al. , viral rn et al. .Section S7. As shown in From 6 hpi to 10 hpi the radical load is decreasing. This decrease might be due to a decrease in virus-producing rate or cells gradually losing their ability to produce radicals at a high viral load. Also the radicals produced in 6 hpi might be gradually transformed into non-paramagnetic ROS. This is also supported by the DCFDA assay (to measure both paramagnetic and non-paramagnetic ROS) in UV irradiation can damage the genome of the virus, which will inhibit viral replication. However, not only the viral replication but the viral components can also induce NO* overproduction. As a result the cell's response to UV-inactivated SFV-FND conjugates is similar to infectious SFV-FND. A similar response is seen also in inactivated viral vaccines where cells also respond without the actual infection. As a comparison, the control group (BHK-21 cells with SA-FND) did not show any significant changes . The sam3.71 measurement cycle for 85,000 times in 85 min. Then we deployed the “moving window algorithm” to analyze the collected data to investigate the pseudo real-time evolution of the T1 relaxation which is explained in 1 measurement cycles for quantifying the T1 value, a small subset was used for quantifying the T1 to show the rolling average. Therefore, this analytical method does not necessarily improve the temporal resolution in a conventional sense, but it does provide the evolution of T1 as a function of time. After probing the intracellular free radical concentration in BHK-21 cells every 2 h post SFV infection and the corresponding FND movement, we evaluated the “pseudo real-time” free radical response of host cells to the intruding virus. In these experiments, we apply the T1 obtained from BHK-21 cells treated with infectious SFV-FND  and during 30–40 min (from 270 to 130 μs) when cells were treated with infectious SFV-FND. This decrease did not occur in the control group. Interestingly, these changes in radical load are occurring during the fusion and uncoating period in the early SFV infectious process . Moreover, stress granules (SG) formation occurs transiently after viral uncoating but before viral mRNA transcription to shut the host protein synthesis off [1 at 10–15 min can probably be attributed to the fusion event, while the decreasing T1 at 30–40 min is probably due to the SG formation after fusion and uncoating. The variations were also shown in inactivated SFV-FND treated cells, but with less amplitude comparing the red and green curve in SFV-FND conjugates were used to investigate the radical response at the place of virus entry. The variation in TD green, b–c is siD green, b–c. We ome scale e. Moreovesis off ,51. One esis off . As a reViral membrane fusion and uncoating are interesting processes in the early-phase of infections. Widely-used methods to detect this processes are electron microscopy ,54 and f3.81 relaxation of 50–100 nm FND is affected by pH. In fact, the authors proposed a diamond magnetometry based pH-metry to measure pH changes in the range of 3–11. On the other hand, virus infection has been shown to alter the intracellular [1 we conclude that T1 was not influenced by pH significantly in the relevant pH range [Last year, Fujisaku and co-workers demonstrated how the Tcellular ,56 and ecellular  pH by upcellular ,58, whetpH range .1 inside a cell due to the viral infection. Hence, their effect on T1 observed during our measurements can be disregarded. Furthermore, it has been shown that ROS is often involved in the signaling pathway while cells cope with the photo illumination [1 data is to be acquired over longer durations. In fact, as certain cell types are more susceptible to phototoxicity than others [1 measurements depending on the experimental design . However, artifacts generated due to photo-induced free radical formation were excluded by including a control sample (where only time was passed and no interventions were made).Similar to pH, temperature also affects the coherence time of the NV centers. The application of NV-based thermometer for the intracellular temperature sensing was also demonstrated . In addimination ,63. Thern others , cellula4Here we have demonstrated the utility of relaxometry to study unprecedented details of the free radical response of host cells upon viral infection. Using SFV and BHK-21 as a model system, we probed the free radical response of the cells until 10 hpi. The general intracellular radical response was investigated by treating host cells with FND and SFV separately, and intracellular radical response near viral particles was investigated by virus-diamond conjugates (SFV-FND).1, which represent the intracellular free radical concentration near viral particles. This is not possible with any other method. Immunostaining was used to determine where we conduct the T1 measurements. Moreover, a pseudo real-time technique based on relaxometry and virus-diamond conjugates was used to detect radical formation during viral membrane fusion and uncoating events from cellular radical response. Obtaining such information with the conventional techniques is impossible.(i)When BHK-21 cells were treated with FND and SFV separately, we found that the free radical concentration in infected host cells does not change significantly. Moreover, we reveal the unique potential of this method to probe the systemic alterations in the cytoplasmic transport. SFV infection was found to elevate the FND movement within the cytoplasm as quantified in terms of maximum particle displacement. (ii) By using SFV-FND, relaxometry is able to monitor the radical variation at the site of viral production during viral replication and even in the early infectious process. We observed alterations of TGoing forward, efforts need to be taken to (1) efficiently track the movement of the FND (2) implement sophisticated pulsing techniques such as double electron-electron resonance measurements for identifying specific free radical species and enhancing specificity and (3) reduce the FND-to-FND variation in its quantum sensing properties. Finally, we hope that this novel and powerful method will enable researchers to discover and/or redefine the fundamentals of the viral infection and thereby contribute to the design of strategies to combat them.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Based on the Digestible Indispensable Amino Acid Score (DIAAS), egg white protein (EGG) has an excellent score, comparable to that of whey protein but with a lower amount of leucine. We examined the effect of EGG feeding on rat skeletal muscle gain in comparison to that of two common animal-derived protein sources: casein (CAS) and whey (WHE). To explore the full potential of EGG, this was examined in clenbuterol-treated young rats. Furthermore, we focused on leucine-associated anabolic signaling in response to EGG after single-dose ingestion and chronic ingestion, as well as clenbuterol treatment. Because EGG is an arginine-rich protein source, a portion of the experiment was repeated with diets containing equal amounts of arginine. We demonstrated that EGG feeding accelerates skeletal muscle gain under anabolism-dominant conditions more efficiently than CAS and WHE and this stronger effect with EGG is not dependent on the arginine-rich composition of the protein source. We also demonstrated that the plausible mechanism of the stronger muscle-gain effect with EGG is not detectable in the mechanistic target of rapamycin (mTOR) or insulin signaling under our experimental conditions. We conclude that EGG may have a superior efficiency in muscle gain compared to other common animal-based proteins. Skeletal muscle mass is moderated by the balance between the rate of protein synthesis and protein breakdown, both of which are highly influenced by dietary protein intake . DiffereWHE is leucine-rich (8.6 g/100 g protein) compared to typical plant-derived soy protein (5 g/100 g protein). Interestingly another animal-derived protein, egg white protein (EGG), contains lower levels of leucine (3.6 g/100 g protein)  but has Therefore, in the present study, we examined the effect of EGG feeding on rat skeletal muscle gain in comparison to that of the common animal-derived protein sources CAS and WHE. To emphasize the potential of EGG, the effect was examined in clenbuterol-treated young rats, which is considered as the condition of being highly anabolism-dominant. We hypothesize that EGG feeding would result in greater muscle gain. Furthermore, we focused on the activation level of leucine-associated anabolic signaling in response to EGG after single-dose ingestion and after chronic ingestion, along with the interaction of clenbuterol treatment. Because EGG is an arginine-rich protein source and arginine functions in an anabolic hormone secretion promoter ,22, a poThis research was approved by the Animal Studies Committee of Niigata University of Health and Welfare . Male Wistar rats (4 weeks old) were obtained from CLEA Japan . Rats were housed individually at constant room temperature (23 ± 1 °C) in a 12-h light (06:00−18:00 h) and 12-h dark cycle and were provided standard laboratory chow  and water ad libitum until the experiment commenced. After an acclimatization period of one week, all rats were used in the experiment.The exact composition of each protein source is shown in Rats were randomly assigned to either a fasting, CAS, EGG, or WHE group . After 2Rats were randomly assigned to one of the CAS, EGG, or WHE groups, and were maintained on Diet 1 and clenbuterol treatments for 3 or 7 days, as described above, except for saline or clenbuterol administration . On day Rats were randomly assigned to either a CAS, EGG, or WHE group, and were fed the arginine-modified Diet 2 containing the respective protein source . They weRats were randomly assigned to one of the CAS+saline, CAS+clenbuterol, EGG+clenbuterol, or WHE+clenbuterol groups, and were maintained for 2 days on Diet 1 for feeding and clenbuterol treatments as above . On day Rats were randomly assigned to one of the CAS+saline, CAS+clenbuterol, EGG+clenbuterol, or WHE+clenbuterol groups, and were fed Diet 1 of the respective protein source ad libitum for 7 days . During 2, 1 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 2 mM PMSF, aprotinin (10 μg/mL), and leupeptin (10 μg/mL). The homogenates were rotated end-over-end at 4 °C for 60 min and then centrifuged at 10,000× g for 10 min at 4 °C. Aliquots of supernatants were used for immunoblot analysis. Briefly, supernatants were electrophoretically separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight at 4 °C with primary antibodies, followed by incubation for 60 min with appropriate HRP-conjugated second antibodies. Primary antibodies against mTOR, phospho-mTOR (Ser2448), Akt, phospho-Akt (Thr308), S6, and phospho-S6 (Ser235/236), cAMP response element-binding protein (CREB), and phospho-CREB (Ser133) were obtained from Cell Signaling Technology . Immunoreactive bands were visualized using enhanced chemiluminescence reagent , and quantified using NIH Image software.Plantaris muscles were homogenized in ice-cold buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgClBlood glucose levels were measured using the product Glutest Every . Enzyme immunoassay kits were used for measuring insulin , growth hormone , and IGF-1 .The measurements of protein content in each protein source and the composition of amino acids in each diet were conducted by Sunatec, food analysis technology center, Yokkaichi, Japan.t-test. A value of p < 0.05 was considered significant.Values are expressed as means ± SE. Differences among multiple groups were determined using a one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test. When two mean values were compared, analysis was performed using an unpaired p < 0.05), and also weaker than both CAS and WHE in S6 (p < 0.05). For blood parameters, no significant difference was observed among groups except insulin (p < 0.05).We first attempted to identify the effect of EGG itself on muscle signaling  and bloo insulin B where Wp < 0.05). Under these conditions, in plantaris muscle from rats fed EGG, we also found a greater muscle-gain effect (p < 0.05). Regarding the soleus muscle, no difference was observed between groups. On day 3, when phosphorylation levels of Akt  but the magnitude was identical among different protein sources. In these muscles, we also found considerable activation of Akt (B), mTOR (C), and S6 (D) in response to clenbuterol administration, with the lowest response observed in the CAS+clenbuterol group. In Akt and mTOR, these phosphorylation levels were significantly higher with EGG+clenbuterol and WHE+clenbuterol than those in the CAS+saline and CAS+clenbuterol groups (p < 0.05) but no significant difference was observed between EGG+clenbuterol and WHE+clenbuterol.To examine whether the stronger muscle-gain effect of EGG was also superior to CAS and WHE under clenbuterol-treated conditions, we tested the acute effect of clenbuterol administration on these signaling molecules . Clenbutp < 0.05). Between EGG and WHE, body weight was greater in the EGG group throughout, but the difference was statistically significant only on day 6 (p < 0.05). The total food intake in the CAS+clenbuterol group was significantly lower than in other groups (p < 0.05).We further examined the effect of chronic EGG feeding under clenbuterol treatment. During the 7-day period, body weights in all groups were gradually increased A. A tendp < 0.05).In general, clenbuterol treatment decreased both epididymal and retroperitoneal fat pad weights . As showp < 0.05). The same tendency was also observed in the soleus muscle (p < 0.05).In contrast to the effect of EGG feeding on fat pad weight, a marked increase was observed in muscle gain . Clenbuts muscle A,B. The p < 0.05) groups when these were measured 20 h after the last clenbuterol administration and the 6-h fasting.To determine the molecular mechanism behind the EGG-induced facilitation in muscle gain, we examined the effect of EGG feeding on the phosphorylation levels of Akt and mTOR signaling molecules . AlthougAside from the negative concept linking egg consumption and the adverse effects of cholesterol, there is accumulating evidence that eggs and egg-derived foods perform positive functions in promoting health . In regaWe also provided a result showing that EGG-induced facilitation in muscle gain is apparent without clenbuterol treatment. Therefore, we focused on the contribution of EGG itself. When EGG was ingested, activation levels of mTOR and S6 were statistically lower than those of other protein sources or WHE 1 h after EGG feeding. Although we did not measure leucine concentration in blood, the level might be lower in EGG than others, and this would be a plausible candidate for lower activation of mTOR signaling. Also, given that insulin is capable of activating mTOR , lower iAlthough EGG has an almost equivalent or lower leucine amount than CAS and WHE, it is known to be an arginine-rich protein source . This hiWhereas young mammals have been used as a good experimental model to estimate the effect of nutrients or food on the whole body or fractional anabolic effect, it should be noted that arginine is categorized as a semi-essential amino acid and is needed for optimal growth in young mammals, but not in adults . This isWe found that EGG feeding resulted in a marked reduction in visceral fat mass under growth and clenbuterol-treated conditions. This is in accordance with previous studies ,18,19 buTo emphasize the potential of EGG and to see whether the EGG-induced facilitation in muscle gain would appear under highly anabolism-dominant conditions, we used clenbuterol. Clenbuterol has been known to be a strong anabolic reagent and it can induce significant muscle hypertrophy within a few days ,30,31. IWe have also provided insight into clenbuterol-induced anabolic effects on skeletal muscle. Clenbuterol has a powerful anabolic effect and appears to cause physiological skeletal mass to achieve maximum efficiency. However, few animal studies have shown clenbuterol treatment to result in an additive anabolic response when combined with muscle activity-associated stimuli/conditions   or recovIn the present study, we had only single sampling points for blood and muscle signal transductions, therefore this is a limitation and a point for further discussion. Nevertheless, our effort to identify the mechanism for the stronger muscle-gain effect induced by EGG on mTOR and insulin signaling might not be a good strategy considering our experimental conditions. Insufficient activation of mTOR signaling after single-dose EGG ingestion has also been reported previously . AdditioAs another limitation of our experiments, we did not measure the effect of EGG feeding on muscle power output, muscle fiber-type compositions, or cross-sectional area. These surrounding results would be convincing proof. In addition, we cannot conclude from the present study whether EGG can induce its stronger muscle-gain effect under conditions that are catabolism-dominant, such as cachexia, malnutrition-associated muscle atrophy, muscle disuse, or sarcopenia associated with these conditions. We hope that these questions can be answered by future research.In the present study, we demonstrated that EGG facilitates skeletal muscle gain under highly anabolism-dominant conditions compared to CAS and WHE and this stronger muscle-gain effect of EGG is not dependent on an arginine-rich protein source. The plausible mechanism of the stronger muscle-gain effect induced by EGG was not detectable in mTOR and insulin signaling under our experimental conditions. We conclude that EGG may have a superior efficiency in muscle gain compared to other common animal-based proteins.


A woman’s ability to achieve her preferred family size is critical in addressing issues of high fertility in sub-Saharan Africa. The socio-cultural context in sub-Saharan Africa presents some difficulty for the attainment of preferred fertility for many women. Few studies in sub-Saharan Africa have examined the extent to which women are unable to achieve their preferred family sizes. This study, therefore, examines the factors that are associated with the non-attainment of women’s preferred fertility by the end of their reproductive years.The study analyzed pooled cross-sectional data with a sub-sample of 1,888 currently married women aged 45–49 years from five rounds of the Ghana Demographic and Health Survey, 1993 to 2014. Test of associations and multinomial logistic regression analysis were used to examine the predictors of underachieved and overachieved fertility relative to achieved fertility.The results indicate that 44 per cent of the women recorded overachieved fertility while about 36 per cent underachieved their fertility. Partner wants more, experiencing child loss and married more than once were significantly associated with overachieved fertility. Nonetheless, increased years of a woman’s education and delaying her at first birth were negatively associated with overachieved fertility. On the other hand, underachieved fertility was significantly associated with having a partner with fewer fertility preference, being of the Islamic faith and ever use of modern contraception.Partner’s fertility preference, child loss experience, marrying more than once and ever use of modern contraception were important predictors of a woman’s inability to achieve her fertility preference. Policies to regulate men’s fertility behaviour, delaying age at first birth, use of modern contraception, encouraging longer years of education, and reducing infant and child mortality are important strategies to achieve fertility preference in Ghana. Fertility preference, which describes how many children a woman would want to have during her reproductive life, is lower in developed countries compared to that in developing countries , 2. It hOur interest in investigating the observed fertility gaps of older women who have completed their childbearing cycle within the sub-region is influenced by the observed stalling of Ghana’s fertility rate in the recent past, which defies our understanding of the demographic transition theory and the resurgence of research interest in fertility desires, family size intentions and realization among various age groups across the globe in recent years –14. HoweIn Ghana, whilst there was rapid decline in fertility between 1988 and 1998, fertility was stalling between 2003 and 2014. Total fertility rate (TFR) declined from 6.4 in 1988 to 4.6 in 1998 –19, i.e.Ghana has made significant progress in socio-economic development over the past decades. For example, 23 percent of the population above six years had never attended school in 2010, compared to 73 percent in 1960 . The extNotwithstanding the cultural context of sub-Saharan African women that makes the attainment of a woman’s preferred fertility quite difficult, it is not so for all women. Yet, few studies have examined the factors that enable some women to actualize their preferred fertility in these socio-culturally high fertility settings in SSA. However, among the few studies that have examined differences between preferred and actual fertility, only few have simultaneously studied factors associated with underachieved and overachieved fertility among women who have completed their fertility –26.In the light of the foregoing, this paper more specifically examines the relationship between fertility preference and actual fertility among women 45–49 years (who have completed their reproductive cycle) in Ghana, which is conceptualized as the fertility gap in the context of the child loss experiences of women. It is conceived that ordinarily, women with high fertility preference would accordingly have equally high actual fertility and women who indicate low fertility preference would correspondingly record low actual fertility with all things held constant. However, this reasoning is dependent on the biological capability of the women  to give birth in accordance with their preconceived fertility desire. At the same time, the realization of this desire could be affected by the child loss experiences of the women, which could shift a woman’s progression to her preferred fertility to be lower or higher, thereby creating a fertility gap between preferred and actual fertility. The limitation underlying this assumption is that the fertility preference question in the Ghana Demographic and Health Surveys (GDHS) is asked to women not before they began childbearing, but at their current ages at the time of the survey that brings in the issue of ex-post rationalization: women who already may have had more children at the time of the survey being more likely to rationalize their higher actual fertility by giving responses that would suggest a higher preferred fertility and vice versa.Furthermore, we recognize that while for some women their fertility preference could remain fixed, for many others especially in the context of many developing countries, fertility preference could change in the course of childbearing , 14. ForIn the light of all these circumstances and limitations of the data being used for this study, we further hypothesize that women who would prefer lower fertility may end having higher actual fertility after experiencing child losses to mortality. Conversely, women who may prefer higher fertility may have lower actual fertility in the absence of child losses through deaths and/or having stable marital unions. In either of the two scenarios, a fertility gap is respectively created leading to overachieved fertility in the first instance and underachieved fertility in the second scenario. Sandwiched between these two are other women who would not experience instability in their fertility preferences irrespective of their child loss experiences, resulting in achieved fertility. Implied in these assumptions is that child loss experiences of women alongside other characteristics of the women and their partners could contribute towards our explanation of the fertility gap between preferred and actual fertility, a subject which has not attracted much research attention in Ghana.The rationale for choosing women aged 45–49 years is that fertility gap provides a true reflection of the completed fertility of these women. This study contributes to the limited literature on fertility gap  among women with completed fertility in sub-Saharan Africa by examining the predictors of underachieved and overachieved fertility in Ghana. Studying why women are unable to achieve their preferred fertility provides an opportunity to understand the unique characteristics associated with these women especially the role of child loss experiences of women which have implications for policy intervention.Various theoretical and conceptual frameworks have been postulated in studying fertility preferences, fertility intentions and fertility desires. These include the theory of planned behaviour , the claThis theory has been heavily criticized as considering fertility preference as being fixed or static , 16, 17.There are other women’s socio-economic and partner characteristics that could directly influence actual fertility either positively or negatively. These include rural-urban residential differences, education, ethnicity, religion, and number of marital unions , 36. ForFactors that determine the difference between preferred fertility and actual fertility that is conceptualized as fertility gap in this paper include child loss experience, type of place of residence, years of schooling, ethnicity, religion, age at first birth, use of modern contraceptives, number of marital unions and partner characteristics such as partner’s educational attainment and fertility preference , 21. Ferhttp://dhsprogram.com/data/available-datasets.cfm. The Ghana Demographic and Health Survey (GDHS) is a cross-sectional, nationally representative population-based survey of households, targeting the reproductive lives of women, age 15–49 years and men of 15–59 years.We sought permission and accessed the data from MEASURE DHS database at The GDHS uses a two-stage sampling method made up of cluster sampling, followed by systematic sampling at the household within each cluster. The GDHS sample is, therefore, stratified according to the administrative regions as well as rural-urban classifications using a probability proportional to population size at the initial stage of selection. The study data are based on a subsample of women aged 45–49 years from the various rounds of the GDHS conducted between 1993 and 2014. Though Ghana has conducted six rounds of the demographic and health survey, that of 1988 was excluded as it did not have questions on spousal fertility preference, which was important for this analysis. The study pooled data from the five rounds of the GDHS for 1993, 1998, 2003, 2008 and 2014 drawing on the women’s dataset. Pooling together different cross-sectional surveys increased the sample size and resulted in an increase in the precision of parameter estimates which allow for more precise conclusions to be drawn especially on the sub-population of women age 45–49 years who have completed their reproductive lifespan and may not be sufficiently represented by a single round of survey for such analysis , 41.The pooled dataset from the successive surveys was of great benefit to the study as each survey gives independent estimates of the same sets of measurement and relationships  and eachThe dependent variable used in the analysis in this study is fertility gap. This was measured by subtracting children ever born from preferred fertility of the woman (number of children preferred). The GDHS measures fertility preference by asking women with children: “If you could go back to the time you did not have any children and could choose exactly the number of children to have in your whole life, how many would that be?” This question allows both numeric and non-numeric responses. The non-numeric responses include “Up to God,” and “Don’t Know”. Women who provided non-numeric responses (146 cases) were dropped from the study. The reason for the exclusion of non-numeric responses from the analysis is that studies suggest that women with numeric responses make predictive outcome of child preferences compared to those with non-numeric responses . BesidesThe difference between preferred fertility and actual number of children ever born yielded three different outcomes: negative, zero and positive values. The negative values constituted overachieved fertility, the zero value was labelled as achieved fertility and the positive values greater than zero constituted underachieved fertility.Both the socio-demographic profile of respondent and partner characteristics were examined as possible predictors of fertility gap. Child loss experience of a woman refers to women with children dying within 59 months after they are born either at infant or childhood ages. This variable is categorized into four: all children survived; one or more infant deaths; one or more child deaths; and both infant and child deaths. The other socio-demographic variables include age at first birth ; number of marital unions (once or two or more unions); ever-use of modern contraceptives (Yes or No); respondent’s educational attainment (in years); place of residence ; religion  and ethnicity . The contraceptive schedule of the GDHS covers a range of questions including ever-use and current use of contraceptives. For this study, we did not focus on current use of contraceptives knowing that women within this age group have either ended or are ending their fertility. For this analysis, we selected ever-use of contraceptives to examine whether ever-use of contraceptives influences their achieved fertility and thereby contribute to explaining the variation between actual fertility and fertility preference which constitutes the fertility gap. Hence, it is expected that women who have ever used modern contraceptives may be more likely to have their actual fertility approximating their preferred fertility  compared to those who never used modern contraceptives. Ethnicity was classified based on the lineage system in Ghana where the Akan ethnic group represents the matrilineage while Ga-Dangbe and Ewe to the south and Mole-Dagbani represent patrilineage. Partner characteristics include the partner’s level of education (in years) and his fertility preference .2) and one-way analysis of variance were used to test association between fertility gap and each of the explanatory variables at a p-value of p < .05. At the multivariate level, the multinomial logistic regression model was used to examine the net influence of possible individual socio-demographic and spousal characteristics on underachieved and overachieved fertility compared to achieved fertility among currently married women, age 45–49 years who have completed their reproductive cycle. Women with achieved fertility were used as the reference category because it was assumed that women’s actual fertility should equal their fertility preference in an ideal situation notwithstanding the fact that in sub-Saharan Africa the socio-cultural contexts present some difficulty for many women to attain their fertility preference. We, therefore, seek to understand what influences women to deviate from their expected fertility preference to realize either underachieved or overachieved fertility compared to others who are able to achieve their fertility preference. In other words, achieved fertility is considered as the reference group (base category) because it is considered as the ideal expectation although not the most frequent outcome of fertility gap in sub-Saharan Africa, and the two other outcome categories (underachieved and overachieved fertility) are estimated relative to achieved fertility. The model predicted underachieved and overachieved fertility among women who have ended their reproductive lives. The general multinomial logistic regression model that was used is as follows:Descriptive techniques of analysis including the use of percentages were used to describe the differences among the women by fertility gap. Chi-square test  and ‘o’ (overachieved fertility) in the equation indicate which outcome the parameters belong to. The equation for the log of the relative risk ratio has two equations and a constraint, where:o, β1…βn are regression coefficients.βThe advantage of the multinomial is that it provides a way to explore the lack of symmetry between underachieved and overachieved fertility. At the multivariate level, using STATA, coefficients from the pooled data describe the relationship between the covariates and fertility gap.The main limitation is that the survey question on fertility preference derived from the desired family size question is prone to the challenge of ex-post rationalization. This is because respondents may not include possible child deaths in their ideal family size. In addition, preferred fertility is influenced by individual and household circumstances at the time of the survey. This could lead to respondents upwardly giving biased responses. Childhood mortality experience of women is one of the socio-economic variables in this study to address the problem of ex-post rationalization. Furthermore, findings from the survey could suffer from a time-sequence challenge in determining factors associated with fertility gap, hence, it is difficult to draw clear causality conclusions. Despite these limitations, this study is one of the few to examine the predictors of underachieved and overachieved fertility among women with completed fertility in the context of sub-Saharan Africa. Furthermore, the findings of this study provide policy makers with a clearer understanding of factors that need to be considered to further reduce fertility.The results showed that of the 1,888 sub-sample of women, age 45–49 years, a higher proportion of women (44.0%) with completed fertility had recorded overachieved fertility, while 20.1% of them had achieved fertility with nearly 36 percent (35.9%) having underachieved fertility .Women with higher mean number of children ever born reported having overachieved fertility . There wUnderachieved and achieved fertility was highest among women who had first birth at later ages (21 years and above). More than half (57.8%) of women who have completed their reproductive cycle with first birth before 18 years had overachieved fertility.The number of marital unions was significantly associated with fertility gap. While underachieved and achieved fertility was highest among women who had been in one marital union (39.5% and 23.4% respectively), overachieved fertility was highest among women who had been in two or more unions.Ever-use of modern contraceptives was significantly associated with fertility gap. The results in Partner’s fertility preference was significantly related to fertility gap. Underachieved fertility was higher among women whose partners want fewer children compared to others whose partners desire to have more children. Concerning achieved fertility, almost a quarter of women (23.6%) whose fertility preference was the same as their partners were associated with achieved fertility. On the other hand, close to half (44.8%) of women whose partners desired higher fertility than them overachieved their fertility preference.Education also emerged from the analysis as a statistically significant predictor of fertility gap where a woman with increased number of years of schooling were less likely to have attained overachieved fertility by 3.8%  compared to achieved fertility. Thus, women with higher number of years in education were significantly less likely to have overachieved fertility by the end of their reproductive lives.Regarding ethnicity, women belonging to the Mole-Dagbani ethnic group were less likely to have attained overachieved fertility compared to achieved fertility by 50% . Similarly, women belonging to the Other ethnic group (predominantly ethnic groups in northern Ghana) were less likely to have attained overachieved fertility with respect to achieved fertility by 32%. This suggests that women belonging to patrilineal ethnic groups were significantly less likely to have overachieved fertility compared to achieved fertility.Women who experienced first birth at the age of 21 years and above were less likely to attain overachieved fertility compared to achieved fertility by 43% . This means that women who delayed first birth were significantly less likely to have overachieved fertility with respect to achieved fertility.The number of marital unions of a woman was a statistically significant predictor of fertility gap. Women who had been in two or more marital unions were more likely to have underachieved fertility compared to achieved fertility by 1.9 times . Similarly, those who had been in two or more marital unions were more likely to have overachieved fertility compared to achieved fertility by 1.9 times .It is to be noted that the results relative to the use of modern birth control methods were contrary to expectation. Women who have ever used modern contraceptives are more likely to have overachieved fertility compared to achieved fertility by 1.4 times . This could indicate that women use contraception when they perceive a risk of overachieving fertility or have already overachieved fertility.The results further show that partners’ fertility preference is a significant predictor of fertility gap. A woman with a partner who wants more children than her was 1.6 times more likely to have overachieved fertility compared to achieved fertility . Again, a woman who does not know the partner’s preference was 1.4 times more likely to have overachieved fertility relative to achieved fertility . The results from Understanding the preferred and actual fertility nexus sheds light on why some women are unable to achieve their preferred fertility and why others are able to do so . HoweverThe study examined the predictors of underachieved and overachieved fertility among currently married women aged 45–49 years in Ghana between 1993 and 2014. This study identified nine factors that predicted overachieved and underachieved fertility: child loss experience, education, ethnicity, religion, age at first birth, number of marital unions, ever-use of modern contraceptives; partner’s fertility preference and year of survey. Five factors predicted both underachieved and overachieved fertility: child loss experience, ethnicity, number of marital unions, ever-use of modern contraceptives and partner’s fertility preference.The results indicate that a higher proportion of women who had all their children surviving child deaths achieved fertility. Achieved fertility was less likely to be attained among women with some child loss experience. This is consistent with what is found in the literature , 45. ThiRegarding education, the study found that higher number of years of education increases the likelihood of underachieved fertility and reduces the likelihood of overachieved fertility. Several studies in sub-Saharan Africa have also identified education as an important panacea to reducing fertility or achieving a woman’s preferred fertility , 48. EduResearch has shown that the likelihood of achieved fertility occurring is higher when first birth is delayed to later ages. This is expected because early age at first birth may predispose most women to subsequent births especially when the births push them into marriage where they may experience longer reproductive years, which could expose them to a higher risk of subsequent births. In contrast, later age at first birth will tend to shorten the reproductive years of women and reduce their fertility in the long run.Furthermore, compared to being in one stable union, being in two or more unions increased the relative likelihood of having overachieved fertility than achieved fertility by 1.9 times. Similarly, being in two or more unions increased the likelihood of underachieved fertility than achieved fertility. This suggests that a woman who has been in more than one union in her reproductive life is significantly less likely to achieve her preferred fertility. The finding from this study is consistent with what some studies have shown , 51, 52.We, however, note from some other studies in sub-Saharan Africa that compared to women in one continuous union, women who have ever been in more than one union have lower fertility . An explThe finding that ever-use of modern contraceptives predicted a less likelihood of underachieved fertility and more likelihood of overachieved fertility relative to achieved fertility is quite unexpected because contraceptive use is expected to reduce fertility among women who actively practise contraception. However, from the results presented in The results reported on partners’ fertility preference imply that men’s importance over reproductive decisions is critical in placing reduction in fertility preference and actual fertility in context , 54, 55.The clear trend toward lower incidence of overachieved fertility could be related to decreasing unmet need for family planning in sub-Saharan Africa. A Study by Nyarko et al  using thThis study observes that underachieved and overachieved fertility is not uncommon among women with completed fertility in Ghana. Furthermore, we have shown that overachieved fertility is declining over the years as represented by the five rounds of the GDHS. This could be due to other factors not included in the regression model. Unmet need for family planning is one of them.A partner’s preference for more children predicted an increased likelihood of overachieved fertility. Considering the role played by men in women’s ability to achieve their preferred family size, efforts to reduce fertility in sub-Saharan Africa must consider how to reduce men’s higher fertility preference. We, therefore, recommend that programmes to reduce fertility in sub-Saharan Africa must consider the significant role played by men in fertility decisions in sexual partnerships especially in marital unions. In addition, researchers must consider placing equal emphasis on the fertility behaviour of men in any analysis of fertility among women to be able to properly inform relevant policy interventions towards achieving further fertility reduction in Ghana and the rest of sub-Saharan Africa.The study findings further suggest some research implications that indicate that reproductive goals are increasingly achieved; further research could assess the fertility implications of this important result. Prospective birth interval data could be used to examine the impact of the death of a child on subsequent birth intervals, thereby assessing replacement effects on fertility goals among women in Ghana and elsewhere in sub-Saharan Africa.


To identify the prevalence of infection in the urban area of the capital city of Belém, Brazil, the Laboratory of Virology of the Federal University of Pará implemented, as a public service, serological screening for human T-lymphotropic viruses 1 and 2 (HTLV-1/2) infection and, if necessary, counseling service and referral to specialized medical care. The project is funded by the National Council of Science and Technology, the Ministry of Health of Brazil and the Pan American Health Organization.® HTLV I/II Score, Fujirebio, Japan) and/or by real-time polymerase chain reaction. G and Fisher's exact tests were applied to identify the association between epidemiological characteristics and HTLV-1/2 infection.From January 2020 to June 2021, 1,572 individuals of both sexes were approached to answer a questionnaire and were tested using an enzyme immunoassay . Seropositive samples were confirmed as HTLV-1 and HTLV-2 infection by line immunoassay (INNO-LIAp = 0.0442). The infected individuals were referred to a counseling session with a nursing professional, and two patients who manifested signs and symptoms suggestive of myelopathy associated with HTLV were referred to a neurologist.Of the 1,572 screened individuals, 63.74% were females between the ages of 30 and 59 years (49.04%). Infection was confirmed in six individuals (0.38%), among whom three (0.19%) were infected with HTLV-1 and three with HTLV-2 (0.19%). Blood transfusion before 1993 was the main risk factor associated with the route of exposure to the virus (The implementation of the screening service revealed the occurrence of moderate endemicity of HTLV-1/2 in Belém, reinforcing the importance of continuing the service as a means of establishing an early diagnosis and providing counseling as a measure to prevent and control viral transmission in the general population. Retroviridae, subfamily Orthoretrovirinae, and genus Deltaretrovirus (Human T-lymphotropic viruses 1 and 2 (HTLV-1 and HTLV-2) belong to the family trovirus . HTLV-1/trovirus , 5.Although most individuals infected with the virus show no symptoms, HTLV-1 is associated with serious diseases, such as HTLV-1-associated myelopathy (HAM) and adult T-cell leukemia/lymphoma (ATL), pathologies that may occur in ~2–3% of people living with HTLV (PLHTLV) , 7. HTLVThe diagnosis of infection is initially performed using an enzyme-linked immunosorbent assay (ELISA) as a screening method for the detection of anti-HTLV-1/2 antibodies. Positive cases are referred for infection confirmation by Western blot, INNO-LIA and/or conventional (PCR) or real-time (qPCR) polymerase chain reaction assays . The firGlobally, it is estimated that 5 to 10 million individuals are infected with HTLV, with the distribution varying based on ethnic group, behavioral risk factors for infection and geographic location; southwestern Japan, sub-Saharan Africa and South America have the highest endemicity of the virus . In BrazPrevious studies conducted in the state of Pará have reported high prevalence rates of HTLV infection in specific groups, such as blood donors , 19, preIt is of paramount importance the counseling of asymptomatic HTLV-1-infected subjects, to perform a clinical follow-up of the individual aiming to detect early the symptoms of diseases associated with the virus. Additionally, the lack of public policies for the active identification of cases of infection and subsequent counseling and screening for intrafamilial transmission results in a loss of productivity for those affected and their families. In addition, the absence of policies for preventing new infections and for providing adequate care for those living with HTLV-1 contributes to the increase in socioeconomic disparity between and within countries . Given tIn the period from January 2020 to June 2021, the Laboratory of Virology of the Federal University of Pará began the HTLV-1/2 serological screening project in the city of Belém, the capital of the state of Pará, and tested a total of 1,572 individuals of both sexes who were categorized into the following age groups: children (7 to 11 years); adolescents (12 to 17 years); adults (18 to 59 years); and elderly individuals (>60 years).via the promotion of social actions in health, carried out primarily in community and religious centers in different neighborhoods in the capital. A portion of the sample consisted of people who voluntarily sought the Laboratory of Virology of UFPA, seeking a diagnosis for an infection, after extensive posts and dissemination on social networks and in a newspaper (printed and online) with high circulation in the state.The individuals were approached through an active search The participants were given basic information about HTLV and educated on the importance of diagnosing the infection and implementing prevention methods. All participants answered an epidemiological survey, after which peripheral venous blood was collected into 4-mL EDTA tubes. Subsequently, the samples were centrifuged to separate the plasma and cells (erythrocytes and leukocytes) and stored at −20°C until serological and molecular tests were performed at the UFPA Laboratory of Virology. The participants were informed that if they presented reactive or indeterminate results, blood would be collected again to confirm the infection and that if infection was confirmed, they would be referred to counseling and follow-up with a nurse. Individuals who manifested clinical signs suggestive of diseases associated with HTLV were referred to specialized medical care.Plasma detection of total anti-HTLV-1/2 antibodies was performed using an ELISA  following the manufacturer's protocol. Samples with reactive or indeterminate results (cut-off = 0.284) were subjected to confirmatory tests (INNO-LIA and/or qPCR).® HTLV I/II Score kit  was used to detect anti-HTLV-1 and/or HTLV-2 antibodies following the manufacturer's protocol.LIA was used as a supplementary confirmation method for HTLV infection and used to differentiate viral types. An INNO-LIAFor the analysis of HTLV proviral DNA, DNA was extracted from 200 μL of cell mass (leukocytes) with a QiaAmp DNA mini kit  following the manufacturer's protocol.pol (186 bp) of HTLV-1 and tax (75 bp) of HTLV-2 were used as viral confirmation and molecular differentiation targets  on the Applied Biosystems StepOne Plus Real Time PCR platform. The human albumin gene was used as an endogenous reaction control, and the viral gene regions  targets . Each re-3′ (HTLV-2R), 5′-GCTGTCATCTCTTGTGGGCTGT-3′ (Albumin F), and 5′-AAACTCATGGGAGCTGCTGGTT-3′ (Albumin R). The probe sequences were as follows: FAM-5′- CTTTACTGACAAACCCGACCTACCCATGGA-3′-MGB (HTLV-1), FAM-5′- TGTCCCGTCTCAGGTGGTCTATGTTCCA-3′-MGB (HLTV-2) and FAM-5′- CCTGTCATGCCCACACAAATCTC-3′-MGB (Albumin) (The following primers were used in the reactions: 5′-CCCTACAATCCAACCAGCTCAG-3′ (HTLV-1F), 5′-GTGGTGAAGCTGCCATCGGGTTTT-3′ (HTLV-1R), 5′-CGATTGTGTACAGGCCGATTG-3′ (HTLV-2F), 5′-CAGGAGGGCATGTCGATGTAGAlbumin) .p < 0.05).The data obtained from the epidemiological survey answered by the participants was input in a database in Epi-Info 7.2. Statistical analyses were performed using BioEstat 5.3  and GrapThis study complied with ethical precepts and was submitted to and approved by the Human Research Ethics Committee of the Health Sciences Institute of UFPA and by the National Research Ethics Committee , in accordance with Resolution no. 466/12 of the Ministry of Health of Brazil. Individuals over 18 years of age who agreed to participate signed an informed consent form. Children under 18 years of age signed an informed assent form, and their legal representatives signed an informed consent form, authorizing their participation in the study.Of the 1,572 individuals evaluated, 63.74% were female, with a mean age of 41 years, ranging from 30 to 59 years (49.04%). Among the other characteristics identified in the epidemiological survey, 54.03% self-reported being mixed race, 33.81% reported having a higher education, and 38.69% reported having a family income equal to or greater than five times the minimum wage, with no significant differences between infected and non-infected individuals .After obtaining the serology results for anti-HTLV-1/2 antibodies, six individuals were positive and were contacted by telephone to schedule an appointment for blood collection to confirm the viral infection. The new samples were subjected to the same testing sequence, and ELISA and INNO-LIA were performed again. Additionally, qPCR was performed as previously described. Infection was confirmed in six individuals , with 3 (0.19%) infected by HTLV-1 and 3 infected by HTLV-2 (0.19%). Among those infected, 66.7% were women aged 49 to 87 years .INNO-LIA indicated two cases of an HTLV profile without defining the viral type, and qPCR confirmed the presence of proviral DNA of HTLV-1 and HTLV-2. One HTLV-1-positive individual, as determined by INNO-LIA, did not have proviral DNA detected by qPCR .When evaluating the responses obtained in the epidemiological survey answered by the six HTLV carriers, blood transfusion was the main risk factor associated with the virus exposure route (p = 0.0442) . HoweverThe infected individuals received their respective laboratory diagnostic for the infection and were referred for a first counseling session with a nurse who took the medical history of the patients and talks about some virus' aspects, such as transmission modes, diseases associated, intrafamilial segregation, prevention, and treatment. The close relatives  of the six patients were invited to undergo testing for possible intrafamilial transmission; however, to date, they have not yet responded to our first call.Of the six individuals with confirmed cases, four were asymptomatic. Two female HTLV-2-positive individuals had clinical signs suggestive of neurological disorders and were referred for evaluations by a neurologist, as described below.#04 (HTLV-2), female, single, 49 years old, had incomplete primary education and reported in the survey that she had been pregnant three times and breastfed for more than 6 months. At the time of the interview, she reported not having an active sex life. During counseling with a nurse, she reported feeling hand tremors, urinary urgency, swelling and joint pain, with restricted movements and gait changes.Patient #06, widower, 87 years old, was hospitalized during the study with severe pulmonary fibrosis and presented with alternating consciousness, which prevented a complete neurological evaluation. It was possible to observe only the absence of clear signs of HAM, but there was a history of urinary incontinence associated with motor deficit (leg weakness) but without signs suggestive of a pyramidal corticospinal tract injury.Patient Investigations of the prevalence of HTLV-1/2 infection in the state of Pará have been conducted in specific population groups, such as blood donors, indigenous populations, the Quilombola people, PLHIV and Japanese immigrants , 28–30. A study by Silva et al. , conducttax gene), low quality of extracted DNA, mutations and a low proviral load, which can potentially lead to the absence of gene amplification and thus result in a false-negative diagnosis for infection. Therefore, the use of a single laboratory method to confirm and differentiate HTLV-1/2 infection is not recommended; it is necessary to adopt complementary confirmatory assays to minimize diagnostic and prevalence estimate errors.During the screening and confirmation analyses, two individuals were identified by INNO-LIA with a serological profile of HTLV, and qPCR molecular analysis confirmed the presence of HTLV-1 proviral DNA in one individual and HTLV-2 proviral DNA the other. Another individual was positive for HTLV-1 based on INNO-LIA diagnostic criteria, but proviral DNA was not detected by qPCR. These serological (INNO-LIA) and molecular (qPCR) methods have been used and endorsed for the confirmatory and differential diagnosis of HTLV-1 and HTLV-2 infections . DiffereIn addition to an accurate laboratory diagnosis that allows identifying the prevalence of infection in a given geographic area, it is necessary to implement a counseling and follow-up service for PLHTLV to provide efficient clinical, laboratory and psychological monitoring. Rosadas et al.  emphasizThe results presented here are part of the first year of a screening, confirmation, counseling and multiprofessional follow-up service for PLHTLV offered through the Laboratory of Virology of the Federal University of Pará and implemented as part of a National Network of laboratories, funded by the Ministry of Health and PAHO. The main limitation of the study can be considered the difficulty of convincing the seropositive individual to perform a new blood collection in order to carry out the confirmatory tests. On the other hand, the main strength of the study is to contribute with the prevention and control measures for HTLV infection to reduce transmission and to identify the main risk factors associated with infection, characterizing the individuals or groups that may be more vulnerable to HTLV, and thus provide information that will support the development of public health policy strategies to combat the spread of this virus in Brazil.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Human Research Ethics Committee of the Health Sciences Institute of the Federal University of Pará and by the National Research Ethics Committee (CAAE No. 27290619.2.0000.0018). The patients/participants provided their written informed consent to participate in this study.AV and IC conceived and designed the study. FL, MV, AL, and FF performed the laboratory experiments. FL, RS, JC, MV, AL, RF, AR, and LM performed the fieldwork. CA-A and EM performed clinical follow-ups. FL and AV wrote the manuscript. All authors read and approved the final version of the manuscript.This study received financial support from the National Council of Science and Technology (CNPq # 442522/2019-3 and CNPQ # 302935/2021-5) and the Ministry of Health of Brazil and the Pan American Health Organization (#SCON2021-00310).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.


Compared with L or M silage, the L+M silage combination improved fermentability, as evidenced by higher LA content and a faster pH drop during the first 7 days of ensiling. With prolonged ensiling time, the combined addition of L and M could increase the count of desirable Lactobacillus, decrease microbial diversity, and inhibit the growth of undesirable microorganism, such as Clostridia, Escherichia, and Enterobacter abundance compared with silage treated with CK, L. or M. Application of L together with M could further improve the silage quality of native grass by altering bacterial community structure. In summary, the addition of lactic acid bacteria and molasses increased the relative abundance of Lactobacillus of native grass silage and improved fermentation quality.Ensiling native grass is an effective method to protect the nutritional quality of forage and alleviate feed shortages in the cold winter of the Inner Mongolian Plateau. To improve the usability of native grass resources as feed in China, the effects of lactic acid bacteria and molasses additions on the microbial population, fermentation quality, and nutritional quality of native grass during silage were investigated. Treatments were a control treatment with no additive (CK), lactic acid bacteria (L), molasses (M), and lactic acid bacteria in combination with molasses (L+M), all of which were stored at ambient temperature (17–28°C) for 7, 14, 30, and 60 days. The results showed that all additives improved nutritional value and fermentation quality with low pH and ammonia nitrogen (NH Stipa grandis P. Smirn) and Chinese leymus [Leymus chinensis (Trin.) Tzvel] dominate typical steppe communities , Chinese Leymus [Leymus chinensis (Trin.) Tzvel.], and Dahurian Bushclover [Lespedeza davurica (Laxm.) Schindl.]. Harvested native grass was chopped to lengths of 2–3 cm. Lactic acid bacteria (L) inoculant and molasses (M) were used as additives for ensiling the native grass. L  was provided by the Institute of Animal Husbandry and Grassland of Japan. M was obtained from Baihui Biological Technology Co. Ltd. . The L inoculant was applied at a level of 106 colony-forming units (cfu) per gram of fresh material (FM), and molasses was applied at 30 g/kg of FM. The chopped native grass was mixed and divided into equal portions for four treatments: no additive control (CK), L treatment (L), M treatment (M), and L + M treatment (L + M). All the additives were mixed homogenously with native grass. In detail, each treated batch was divided into four replicates (one for backup) of 200 g each, which were packed into polyethylene bags and vacuum sealed. A total of 64 bags (4 treatments × 4 ensiling days × 4 repeats) were prepared and kept at ambient temperature (17–28°C). After 7, 14, 30, and 60 days of ensiling, fermentation characteristics, chemical compositions, microorganism counts, and the microbial community were analyzed. Initial fresh forage samples were taken before ensiling.Native grass was harvested at the heading stage from a typical grassland  on the Inner Mongolian Plateau, People’s Republic of China, on July 15, 2020. The dominant species harvested were large needlegrass  contents were determined by oven drying at 65°C for 48 h . The cruEach polyethylene bag was opened on a clean bench. About 10 g of fresh forage or silage was blended with 90 ml of sterilized water, and the extract was serially diluted to quantify the bacterial group in a sterile solution. The numbers of LAB, aerobic bacteria, coliform bacteria, yeast, and molds were measured through the method of 3–N) of the sample. The lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) content of silage was analyzed by high-performance liquid chromatography according to the method of 3–N) content was determined according to the method of Last, the liquid extract was filtered through four layers of cheesecloth and filtered paper. The prepared filtrate was used to analyze the pH values, organic acids, and ammonia nitrogen .The E.Z.N.A.Subsequently, extracted DNA samples were subjected to PCR amplification on bacteria 16S rDNA (V3 and V4 regions). For bacterial amplification, the following primers were used: 341F and 805R. The PCR reactions were conducted using the following program: 3 min of denaturation at 95°C, 27 cycles of 30 s at 95°C, 30 s for annealing at 55°C, and 45 s for elongation at 72°C, and a final extension at 72°C for 10 min. PCR reactions were performed in triplicate 20-μl mixture containing 4 μl of 5× FastPfu buffer, 2 μl of 2.5 mM dNTPs, 0.8 μl of each primer (5 μM), 0.4 μl of FastPfu Polymerase, and 10 ng of template DNA. Agarose gel electrophoresis [2% (w/w)] was used to confirm the PCR products. To eliminate the possibility of false-positive PCR results as a negative control, ultrapure water was used throughout the DNA extraction process instead of a sample solution. AMPure XT beads  were used to purify the PCR products, and Qubit was used to quantify them .The PCR products were sequenced on an Illumina platform  after purification and quantification. Fqtrim was used to perform quality filtering on the raw reads under specific filtering conditions in order to obtain high-quality clean tags (v0.94). Vsearch software (v2.3.4) was used to filter chimeric sequences. QIIME2 was used to calculate alpha and beta diversity, with the same number of sequences extracted randomly by reducing the number of sequences to the minimum of some samples, and relative abundance  was used in bacteria taxonomy. The QIIME2 process was used to examine alpha and beta diversity. Blast was used to perform species annotation sequence alignment, and the alignment databases were SILVA and NT-16S.p < 0.05. The data of high-throughput sequencing were analyzed on the free online platform www.omicstudio.cn.The data was subjected using two-way analysis of variance with the fixed effects of additives, ensiling time, and additives × ensiling time using the general linear model (GLM) procedure of SAS 9.3 software . The effect was considered significant when 10 cfu/g FM, respectively. Fresh native grass molds were below the detectable level.The chemical parameters and microbial compositions by native grass before ensiling are presented in p < 0.05) effects on the chemical composition (except DM) of silage. All additives, except for the CK treatment, significantly decreased (p < 0.05) NDF content of native grass silage. In addition, the L + M silage had significantly (p < 0.05) higher CP contents than other silage at the end of ensiling. The WSC contents in M and M + L silages were significantly (p < 0.05) lower than the CK silage. The NDF and ADF content exhibited a continuous downtrend during the whole ensiling process. The interaction of additive treatments and ensiling days had a significant impact on silage WSC (p < 0.05), but did not affect the silage DM, CP, NDF, or ADF contents (p > 0.05).Effects of additives and ensiling days on chemical parameters of native grass silages are shown in P < 0.05). Compared with the control, all additives significantly (p < 0.05) increased lactic acid (LA) concentration, and decreased acetic acid (AA), propionic acid (PA), and NH3–N concentration in silage. Compared with the other treatments, L + M addition further increased LA concentration and decreased the contents of AA, PA, and NH3–N; butyric acid (BA) was not detected in the native grass silage. The highest LA concentration was recorded in L + M silage, which had a value of 29.94% on the 60th day of ensiling (p < 0.05). The pH values of the additive silages were significantly decreased (p < 0.05) compared with the control during 14 days of ensiling, and L + M silage always maintained a lower pH value during ensiling. Moreover, NH3–N content exhibited a continuous increasing trend during the whole ensiling process, and the content of NH3–N in the L + M treatment group remained low after 14 days of ensiling.p < 0.05). Additive treatment silages had lower (p < 0.05) coliform bacteria content and higher L and yeast numbers compared with the control silage after 60 days of ensiling. Compared with other treatments, the L and yeast contents in L + M-treated silage were significantly (p < 0.05) higher, while the coliform bacteria contents were lower than that of other silages at the end of ensiling. The microbial count of silages exhibited a continuous decreasing trend during the whole ensiling process. Mold counts were not detected in any treatment after 14 days of ensiling.p < 0.05), but did not affect Reads, OUT, and Chao1 (p > 0.05). According to the OTU and Chao 1 indexes, the bacterial community’s richness decreased with increased ensiling time in the CK silage. The Shannon and Simpson indexes were remarkably lower in the M and L + M-treated silages than in the CK silage at days 7 and 14 of ensiling (p < 0.05). In addition, the Chao 1 index and Simpson index showed trends similar to the trend in OTUs. Compared with other treatment groups, L + M addition resulted in the decrease in the richness of the bacterial community without dose effect in silage at day 60 of ensiling (p > 0.05). The lowest Shannon index of bacterial diversity was observed in the L + M-treated silage .The bacterial diversity of native grass silage was discovered using high-throughput analyses targeting variable regions 3 and 4 of 16S rDNA. The valid sequences of all 51 triplicate samples added up to 1,294,499 after unqualified sequences were removed. The average Good’s coverage for all of the samples was greater than 99%, indicating that the sequencing depth was sufficient for reliable bacterial community analysis . The intThe PCoA analysis  showed tProteobacteria (89.82%) and Firmicutes (9.87%) dominated the majority of the total phylum sequence in the fresh sample. During the process of ensiling, Firmicutes and Proteobacteria dominated in all groups, but the community composition was affected by the ensiling treatments. Compared with the CK treatment, Firmicutes increased and Proteobacteria decreased in the L and L + M-treated groups. However, the relative abundance of Proteobacteria in M treatments was higher than that of the other treatments. In addition, the increase in Firmicutes abundance was greater with added L + M, and the relative abundance of Firmicutes (at 60 days) was higher.The dynamics of bacterial communities in native grass silage at the phylum level are shown in Pantoea and Pseudomonas, followed by Erwinia. Their abundance decreased greatly during the ensiling process, especially in the treated silage. Lactobacillus was the most abundant genus in silage with L. plantarum added  after 30 days of ensiling. The addition of L and L + M decreased the abundance of Enterobacter and Escherichia compared with silage with added M alone. In the absence of additives, after 60 days of ensiling, the dominant species were Pantoea and Lactobacillus. Under the influence of the fermentation promoter, there was a slight difference from the CK treatment. The main microbes were Lactobacillus (60.96%) and Enterobacter (11.26%) in the control group, whereas the main genera were Lactobacillus (58.29%), Enterobacter (7.72%), Escherichia (4.00%), and Pantoea (4.17%) in the L group. In the M group, the main genera were Lactobacillus (23.86%), Enterobacter (35.31%), Escherichia (20.12%), and Pantoea (1.74%), and the main genera were Lactobacillus (67.44%), Escherichia (4.85%), and Enterobacter (3.29%) in the L + M combination group.Pantoea was the dominant genus in the fresh native grass (FM) (26.48%). Furthermore, Clostridium_sensu_stricto_12 was the subdominant family in FM, following Pantoea. The additive treatments significantly changed the relative abundance of Lactobacillus, Enterobacter, Escherichia, Pantoea, Pseudomonas, Clostridium_sensu_stricto_12, Erwinia, Klebsiella, and Lactococcus after 60 days of native grass silage. Lactobacillus was the most abundant genus in the L + M-treated silage . Molasses enhanced the growth of Enterobacter and Escherichia.To further reveal the effect of additives on the bacterial community in natural forage silage, one-way analysis of variance bar plots of the genus level among native grass silage groups after 60 days of ensiling are shown in The effects of metabolic pathway changes in bacterial communities were quantified by predicting the functional characteristics of bacterial communities. Therefore, 5 cfu g–1 of FM; it is easier for silage to be successful and preserved  and can metabolize WSCs and LA to produce AA, PA, and other fermentation products  initiate lactic fermentation in the early ensiling process, whereas lactic acid rod (Lactobacillus) play a key role in pH reduction at the later stage  can be found below: YL methodology, visualization, validation, data curation, and wrote the original draft. SD and LS interpreted the data and edited the language. QC wrote, reviewed, and edited the manuscript. GG and ZW conceptualization, acquisition of funding, and writing—reviewing and editing of the manuscript. QL and JH software. YJ conceptualization and funding acquisition. All authors have read and agreed to the published version of the manuscript.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.