To combat pandemics, there is a need for rapid point-of-care diagnostics to identify infected patients and to track the spread of the disease. While recent progress has been made in response to COVID-19, there continues to be a need for point-of-care diagnostics capable of detecting biomarkers—such as antibodies—in whole blood. We have recently reported the development of thermally responsive alkane partitions (TRAPs) for the automation of point-of-care immuno-magnetic assays. Here, we demonstrate the use of TRAPs to enable sample-to-answer detection of antibodies against the SARS-CoV-2 virus in whole blood samples. We report a limit of detection of 84 pg/mL, well below the clinically relevant threshold. We anticipate that the TRAP-enabled sample-to-answer immunoassay can be used to track the progression of future pandemics, leading to a more informed and robust clinical and societal response. More than any other event in history, the COVID-19 pandemic raised awareness about the debilitating lack of diagnostic capabilities in all regions of the world. Early in the pandemic, a lack of testing led to uncontrolled community spread and uncertain levels of disease pervasiveness due to untraced cases. As technology evolved to meet the demands of the pandemic, both viral and serological testing played important roles in tracking those infected. Serological testing is particularly helpful for tracking cases where patients were not tested during the acute infection stage, which can be especially useful in a pre-vaccination stage of a pandemic. Moreover, during the initial vaccine rollout, serological testing can help to validate vaccination technologies. Because access to these serological diagnostic tools can have dramatic effects on the spread of disease, increasing access for the current pandemic and for future pandemics is necessary. To accomplish this, serological diagnostics should be point-of-care such that they can be performed in near-patient settings, such as a physician’s office or walk-in clinic, such that no technical expertise or bulky equipment is required. Specifically, the diagnostic should be sample-to-answer, meaning that it should require no user intervention or precise sample manipulation steps [Traditionally, detecting antibody-based biomarkers requires an enzyme-linked immunosorbent assay (ELISA), which can take hours to perform. Furthermore, ELISA-based diagnostics are confined to central laboratories due to the need to perform precise technical steps. Efforts to transition ELISAs to the point-of-care have most notably resulted in lateral flow immunoassays (LFIAs). In LFIAs, a sample is wicked along an ensemble of membranes, where an immuno-sandwich forms at a specific location when the biomarker is present; a tag on the labeling antibody (often gold nanoparticles) results in a visible line in the readout location. LFIAs work well for non-complex samples, such as a nasal swab. However, to be used with whole blood, the sample must first either be centrifuged to recover the plasma from the blood sample, or the whole blood must be diluted by several orders of magnitude. These additional precise steps preclude the assays from being used at the point of care. Thus, while LFIAs for SARS-CoV-2 antibodies have been published ,4 and soThe field of microfluidics has offered numerous solutions aimed at point-of-care diagnostics ,9. Some For example, in Shin et al., the authors use magnetic beads manipulated by an external magnet to capture viral RNA and transport it from one well to another, following the steps of nucleic acid recovery and PCR amplification . MagnetiRecently, we reported a novel method for automated sample and reagent manipulation using thermally actuated pseudo-valves constructed of higher order alkanes ,22,23,24In the present work, we use this second geometric configuration to construct a sample-to-answer immuno-magnetic assay to quantify antibodies against the SARS-CoV-2 spike protein in whole blood samples. In our assay, a capillary blood sample is added to a cartridge containing several reagent zones, separated by TRAPs . These zUsing the TRAP-enabled system, we show distinguishable fluorescence measurements with a limit of detection of 84 pg/mL and a dynamic range that extends up to 1 μg/mL. In the literature, approximately 1 ng/mL ,27,28 isThe 1 μm streptavidin magnetic beads (Pierce) are prepared by gathering 100 μL 10 mg/mL beads to the side of the tube using an external magnet, aspirating out their buffer, and rinsing them with 200 μL of wash buffer, comprised of 25 mM Tris and 150 mM NaCl . The beads are again gathered to the side of the tube using an external magnet, and the wash buffer is removed. Furthermore, 50 μL 200 μg/mL SARS-CoV-2 biotinylated spike receptor binding domain (RBD) protein (ProSci) is added to the 1 mg washed beads and left to incubate for 1 h at room temperature. Following this incubation, the beads are washed three times by magnetically gathering them to the side of the tube, aspirating out the supernatant, and washing them with 200 μL of wash buffer. On the final rinse, 25 μL of 0.1 M phosphate buffer is added to the mass of beads, resulting in 40 mg/mL magnetic beads coated in SARS-CoV-2 spike RBD protein.3) are 3D printed with Prusament UV sensitive resin from Prusa Research. Once cured, a coverslip  is glued to the open face of the cartridge, covering the channel, and left to dry overnight at room temperature. To ensure a hydrophobic surface, we incubate 423 μL glass water repellent (Rain-X) in the cartridge for 30 min at room temperature. Following incubation, excess glass water repellent is removed, and the cartridges are washed three times with water.The cartridges with channels (3 × 3 × 47 mmEicosane (melt temperature = 42 °C), a higher order alkane, is used to form the TRAPs to separate each reagent layer. First, to construct the assay, the cartridges are held vertically and filled with 50 μL solution containing 5 μM Amplex Red  and 1 mM hydrogen peroxide (Fisher Scientific). To prepare eicosane , it is first melted by placing it in a glass vial on a hot plate at 120 °C. Furthermore, 30 μL of melted eicosane quickly hardens as it is deposited by a micropipette atop the Amplex Red layer. Then, 60 μL 0.1 M phosphate buffer is added, then another 30 μL of melted eicosane, then 60 μL 0.1 M phosphate buffer, then 30 μL of melted eicosane, then 50 μL 100 ng/mL horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibodies (ThermoFisher), then 30 μL of melted eicosane. Finally, in the top zone, 2.5 μL 40 mg/mL magnetic beads coated in SARS-CoV-2 spike RBD protein is added.Whole blood is withdrawn from an exposed vessel at the elbow pocket of swine forelimbs within 15 min after the animal is euthanized. The blood is collected into an EDTA-coated collection tube, which is then repeatedly inverted to mix the blood and EDTA. The tube is then stored at 4 °C. Immediately preceding the experiment, the blood is spiked with varying concentrations (0–1000 ng/mL) of rabbit SARS-CoV-2 spike RBD protein antibodies (ThermoFisher). In addition, 50 μL spiked blood samples are added to the top zone of the TRAP assay. All work performed with animals was completed with approval from University of Maryland, College Park Institutional Animal Care and Use Committee (R-AUG-20–47).Fluorescence measurements are taken by placing a cartridge into a portable fluorescence reader designed by our lab, depicted in Image processing is performed using a MATLAB script that automatically crops an image to a 39 × 39-pixel window. This window only contains the region of interest along a cartridge channel. Fluorescence values reported in this paper are generated by calculating the mean of the set of all grayscale pixel values within the pixel window.The heat required to melt eicosane in each device is supplied via a polyimide heating pad that is adhered to the 3D printed support that holds the sample cartridge in place . Upon pl3) gathers and pulls the beads across the first layer of eicosane into the zone containing HRP-conjugated antibodies. The heater is turned off and the beads are left to incubate for 30 min. After incubating, the heater is turned back on to melt the eicosane layers. Then, an external magnet regathers and pulls the beads across the second layer of eicosane, into one of the zones containing phosphate buffer. Here, the beads are released temporarily in the rinse zone to help promote rinsing. The beads are subsequently magnetically gathered and pulled across the third layer of eicosane, into the other zone containing phosphate buffer, and released temporarily. Finally, the beads are pulled across the fourth layer of eicosane into the zone containing Amplex Red and hydrogen peroxide. The heater is turned off, and the fluorescence is measured by the portable device 10 min after the beads reach the final zone.After the blood sample is added to the top zone of the assay and left to incubate for 30 min, the cartridge is placed on the portable heater to melt the eicosane layers. Once melted, an external neodymium magnet . The beads and antibodies are left to incubate at room temperature for 30 min. The beads are gathered to the side of the tube using an external magnet, the supernatant is aspirated out, and the beads are rinsed with 200 μL wash buffer three times. On the final rinse, the beads are resuspended in 50 μL 100 ng/mL HRP-conjugated anti-rabbit IgG antibodies and left to incubate at room temperature for 30 min. The beads are again gathered to the side of the tube using an external magnet, the supernatant is aspirated out, and the beads are rinsed with 200 μL of wash buffer three times. On the final rinse, the beads are resuspended for 5 min in 5 μL elution buffer, which is comprised of 0.1 M glycine (Sigma-Aldrich) at pH 2. Then, 5 μL of supernatant is collected and added to a 50 μL solution containing 5 μM Amplex Red and 1 mM hydrogen peroxide. Fluorescence resulting from manual bead washing is measured using a Synergy LX plate reader from BioTek . After the eluted sample is added to the Amplex Red/hydrogen peroxide solution, the liquid is moved to a 96-well plate. A fluorescence measurement is taken after 10 min.We have implemented a cap for the TRAP cartridge with a blood collection capillary . To collect blood, the fingertip is cleaned and pricked with a lancet. Then, the capillary touches the blood droplet. The blood is quickly wicked into the capillary tube. Once the capillary is full, the blood is loaded into the cartridge by vertically tapping the device. The capillary cap is then removed and replaced with a closed cap. A video of this process can be found in the Electronic Supplement.We have recently shown that we can use TRAPs as stationary partitions that continually separate reagents but are permeable to magnetic beads, thus enabling magnetic microbeads to transport bound molecules across the partition . Here, w2 cross-section within the cartridge, as we previously established that this geometry is narrow enough for the TRAPs to remain stationary as they are melted. As evidenced by We have chosen to use a channel with a 3 × 3 mmWe aim to use our sample-to-answer assay in a near-patient setting, which requires the entire device to be portable and inexpensive. We have developed a handheld instrument to both heat the TRAPs and to read the resulting fluorescence. The optical components of this instrument are based on our previous work , in whicAfter optimization of the cartridge geometry and assay conditions, we ran the full assay to analyze blood samples spiked with a range of concentrations of antibodies against the SARS-CoV-2 spike protein. Following a 30-min incubation once the whole blood sample is added to allow beads to capture antibodies against the SARS-CoV-2 spike protein, the cartridge is placed in the portable fluorescence reader atop the portable heater. The bead slug is pulled through the immunoassay and into the detection zone as shown in For comparison with gold standard methods, we performed a manual bead-based immunoassay  and used a benchtop plate reader for fluorescence quantification. The whole blood samples were spiked with antibodies against the SARS-CoV-2 spike protein. These samples were incubated with magnetic beads functionalized with SARS-CoV-2 spike proteins. After incubation, the beads were manually washed before incubating with HRP-functionalized secondary antibodies. Following incubation, again the beads were manually washed. Because the beads could not be present when measuring fluorescence in the benchtop plate reader, on the final wash step, the beads were resuspended in elution buffer. Finally, the supernatant was collected and added to a plate containing Amplex Red and hydrogen peroxide. The resulting fluorescence was measured in a benchtop plate reader . Note thThe previous experiments demonstrate that this immunoassay is completely automated without sacrificing performance. However, to be truly point-of-care, assays need to be sample-to-answer, implying that untampered samples such as whole blood must be collected and loaded into the cartridge without any precise manual sample transfers. Thus, point-of-care tests cannot rely on venous blood draws performed by phlebotomists and should instead enable the patient to draw their own sample or enable easy collection at a collection site via nurse, lab technician, or physician’s assistant. We have designed our device to pull a precise volume of whole blood directly from a finger stick into the cartridge via a capillary tube. The capillary tube is built into the cap A, which In this work, we have developed a sample-to-answer assay for the detection of antibodies against the SARS-CoV-2 spike protein in whole blood. We report a detection limit that is below the clinical threshold cutoff to be considered positive for anti-spike antibodies. Fluorescence measurements were taken using a portable reader containing a built-in heater to integrate sample preparation steps into the overall system. Finally, we have also integrated the blood collection step using a built-in capillary tube.Further work will continue to improve our system. Movement of the external magnet will be motorized for full automation. In addition, the portable fluorescence imager can be further miniaturized by replacing the ArduCam (which is useful for prototyping) with a simple photodetector. We also hope to further optimize the assay by reducing the incubation time required in each zone. Additional future work will broaden the applications of the TRAP-enabled magnetofluidic assay. For instance, other antibody biomarkers can be detected by using a different antigen on the magnetic beads. As an example, in this work, we detected the antibody to the spike protein, which indicates exposure to either the virus or vaccine; in contrast, for an assay to indicate exposure to the virus only, one could use the nucleocapsid antigen. More generally, any biomarker molecule—including protein and DNA sequences—can be detected with this point-of-care technology by utilizing the appropriate capture molecule. Finally, this technology can be deployed in the next pandemic or for other urgent point-of-care blood assays to improve accessibility to testing.


A total of 16 domestic pig holdings in the East Anglia region were confirmed as infected over a 3-month period. Obtaining viral genome sequences has since become easier and more cost-effective and has accordingly been applied to trace viral transmission events for a variety of viruses. The rate of genetic evolution varies for different viruses and is influenced by different transmission events, which will vary according to the epidemiology of an outbreak. To examine if genetic changes over the course of any future CSF outbreak would occur to supplement epidemiological investigations and help to track virus movements, the E2 gene and full genome of the virus present in archived tonsil samples from 14 of these infected premises were sequenced. Insufficient changes occurred in the full E2 gene to discriminate between the viruses from the different premises. In contrast, between 5 and 14 nucleotide changes were detected between the genome sequence of the virus from the presumed index case and the sequences from the other 13 infected premises. Phylogenetic analysis of these full CSFV genome sequences identified clusters of closely related viruses that allowed to corroborate some of the transmission pathways inferred by epidemiological investigations at the time. However, other sequences were more distinct and raised questions about the virus transmission routes previously implicated. We are thus confident that in future outbreaks, real-time monitoring of the outbreak  Classical swine fever (CSF) is a contagious febrile disease of pigs that remains a severe socio-economic threat to the global pig industry. The virus has been eradicated from many developed countries, but it continues to cause economic losses in endemic regions. The reintroduction of the virus into disease-free areas has a major impact, as highlighted by the re-emergence of the disease in Japan in 2018 after 26 years of freedom  were confirmed as infected over a 3-month period. The outbreak was controlled by culling of infected and contact premises and vaccination was not applied. The first case (IP01) was confirmed on 8 August 2000. However, substantial illness, attributed to porcine circovirus (PCV-2)-associated disease, had been present since 11 July when weaner animals were introduced from an outdoor breeder unit (IP02) which was, at the time, accordingly thought to have been the origin of the outbreak . A routiIn early September, a cluster of further cases was detected in the south of Suffolk, close to IP01. IP07, an outdoor breeding herd with land adjacent to IP01, was confirmed as infected on 5 September. IP08, which had 5,000 growers and finishers, lies to the north of IP07 and had outdoor growers housed on land contiguous to IP07. IP09 also had outdoor sows, gilts, and boars located close to IP01. It was concluded that lateral local spread infected these premises, possibly due to the scavenging of dead pigs from IP01 by wild animals before CSF was confirmed  were added to 300 μl of lysis buffer  containing 5 U/μl of the endonuclease Omnicleave (Cambio). After incubation at room temperature for 30 min, the samples were homogenized in a Precellys 24 homogenizer in tubes with ceramic beads (CR28) with 3 cycles of 5,000 rpm for 45 s, with 30 s pauses between each cycle. Alternatively, the samples were homogenized using a GentleMax homogenizer and dissociated for 54 s using the manufacturer's program RNA_01.01. The homogenized samples were then clarified by centrifugation at 14,000 × g for 2 min, the supernatant was mixed with 1 ml of the Trizol reagent (Thermofisher), and RNA was extracted according to the manufacturer's instructions.Archived tonsil samples that had been stored at −80°C were available from individual animals from premises IP01-IP05 and IP16, whereas samples from premises IP06–IP13 were stored as pools of tissues from up to 4 pigs. A pooled sample (IP01-7.2) was also available from IP01. No samples were available from IP14 and IP15. To maximize the proportion of sequence data derived from the viral genome, as opposed to contaminating host nucleic acid, tonsil samples (~3–5 mmde novo assembly (IVA v1.0.8 using default settings)  with primers 2250F and 3710R .The analysis of the complete E2 gene indicated that most of the samples contained viruses with identical E2 sequences which, in turn, were also identical to the previously published sequence of the virus isolate CSF0708 obtained from IP03 (JQ441582). However, the samples from IP08 and IP12, which were confirmed positive in September, and IP16, which was the last premise detected 3 months after the first case, had viruses with one (IP12 and IP16) or two nucleotide changes (IP08) compared to the sequence from IP02. Accordingly, the use of the E2 gene would not suffice for in-depth molecular epidemiological approaches (data not shown).Near full-length sequences were obtained for all 15 samples: 12,136 nucleotides encoding the polyprotein region and 349/102 nucleotides of the 5'/3' UTRs, respectively (DDBJ accession numbers LC702327–LC702341). The changes in the E2 gene in SF12, SF16, and SF08 identified by sequencing of PCR product were present in the Illumina generated sequences, confirming that they were not the result of a Phusion polymerase error.The alignment of the sequences encoding the almost full-length genome confirmed that the viruses in all 15 samples were highly similar , with only 5–14 nucleotide changes compared to the sequence from IP02, which was taken as the baseline as it was considered to be the first infected premise . NucleotAlthough virus genome sequences from all infected premises were highly similar, the phylogenetic analysis revealed succinct clusters between the viruses, with sequences from IP05, IP13 IP06, and IP16 forming a separate group, and IP06 and IP16 forming a subgroup therein . In addiPhylogenetic analysis of the sequences (SF01-SF16) obtained from the different infected premises highlighted clusters of related sequences that were supportive of virus transmission routes suggested by epidemiological investigations . The seqvia the transport of breeding sows on 17 July from IP07, which would also explain the higher degree of virus evolution and outside clustering of sequence SF11, or transmission could have occurred via another unidentified contact with any of the earlier infected premises. Sequence information was less informative for clarifying virus transmission routes within the south Suffolk cluster of farms, IP01, IP07, IP08, and IP09, although the molecular analysis is not inconsistent with the theory of lateral spread by wildlife scavenging on carcasses left on IP01 prior to disease confirmation , the Scottish Government, and the Welsh Government projects SE2210 and SE2215.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.


The global objective of this work was to manufacture resistant and durable (water resistant) earth renders with good thermal insulation. For this purpose, a medium plastic clayey soil from Kôdéni (Burkina Faso), constituted by kaolinite (62 wt.%), quartz (31 wt.%), and goethite (2 wt.%), was mixed with dolomitic lime (up to 6 wt.%) to manufacture earth renders. The mineralogical, microstructural, and chemical characteristics of dolomitic lime, as well as the physical , hydric (water absorption test by capillarity and spray test), thermal , and mechanical  properties of the earth renders were evaluated. From these studies, it appears that the addition of dolomitic lime induces the formation of calcium silicate (CSH) and magnesium silicate (MSH) hydrates. These CSH and MSH are mainly formed from the pozzolanic reaction between finely ground quartz and the weak silica of kaolinite in basic media. These formed hydrates improve the physical, hydric, thermal, and mechanical properties of earth renders. This improvement is due to the fact that the formed CSH and MSH stick to the isolated particles of the soil, making them more compact. In view of the good mechanical strength and water resistance, as well as the low thermal conductivity, the elaborated earth renders are suitable for habitats with dry climates, such as the Sahel. In Burkina Faso, as in most developing countries, traditional habitats are still built using vernacular construction materials, such as the raw clayey materials used as earth renders. The use of raw clayey materials to elaborate earth renders for protecting habitats in these developing countries has some advantages, such as their good thermal comfort; however, there are important disadvantages linked to their low mechanical properties and weak water resistance. In order to improve upon the above properties, vegetable fibers, organic binders, and small quantities of mineral binders (cement or lime) are commonly introduced  earth renders with good thermal insulation. This scientific work deals with the influence of dolomitic lime (up 6 wt.%) on the physical and mechanical properties of earth renders manufactured with clayey soil from Kôdéni (western Burkina Faso). The effect of dolomitic lime additions on the erosion resistance, abrasion resistance, and thermal conductivity of earth renders was highlighted, and particular attention was paid to the formation mechanism of calcium silicate (CSH) and magnesium silicate (MSH) hydrates during earth renders manufacturing.Bamogo et al.  previousIn view of obtained results and according to the German standard , KOD claThe lime used in this study is commercial dolomitic lime, and it was supplied by the Coveni company in Bobo Dioulasso (Burkina Faso). 2B4O7) to form a glass bead. The glass bead was dissolved in a nitric acid solution, and the solution obtained was analysed by an ICP-AES device.Chemical analysis of the dolomitic lime was carried out by inductively coupled plasma-atomic emission spectrometry (ICP-AES). A sample of raw material was crushed to particles size less than 80 µm and melted with lithium tetraborate  and graphite back monochromator. The range of analysis was between 5 and 75°, with a step size of 0.04°. The acquisition time of the analyses was 2 s. The analyses were all performed on powders previously crushed to particles size less than 80 µm and placed in a rotating sample holder. Analysis by differential scanning calorimetry (DSC) consisted of monitoring the difference in the heat flow between the two containers—one contained the analyzed sample, and the second contained the thermally inert sample . The heating rate during the analyses was 10°C/min. Thermogravimetric analysis (TGA) consists of recording the variation in mass during a thermal cycle. These changes are related to chemical reactions or the departure of volatile constituents adsorbed or combined in a material. The two techniques are often applied simultaneously on the same apparatus and sample. The DSC-ATG thermograms were recorded with a Netzsch SATA 449F3 Jupiter apparatus, under an argon atmosphere between 30 and 1100 °C. A quantity between 10 and 30 mg of the sample, with a maximum particle size not exceeding 80 µm, was tested.−1, diamond crystal-Perkin Elmer). The device used for the analysis was a Perkin Elmer L125000P Frontier.Infra-red spectrometry on crushed dolomitic lime (particles size less than 80 µm) was carried out on an attenuated total reflectance-Fourier transform infra-red spectrometer  was used to explore the dolomitic lime sample surface, in order to provide information regarding the mineral’s morphology. SEM observation of this sample was carried out with JEOL 6380 LV microscope. Energy dispersive X-ray spectroscopy (EDS) was used to access to the chemical composition of the sample’s explored surface. This analysis was carried out using Brüker X Flash 6/30 detector. The earth rendering mortar’s elaboration was carried out according to the protocol described by Bamogo et al. .3) in two layers, with series of manual shocks of 20 strokes total per layer. The molds were kept in the shade  for 72 h. After demolding, the obtained coatings were kept for 90 days at room temperature , in order to avoid the appearance of cracks. To perform the various tests, three samples of each formulation were developed. This required the formulation of 24 samples of different earth renders formulations. The proportions of the mixtures used for the manufacture of earth rendering mortars are given in the For the manufacturing of the raw clay renders, the KOD raw material was oven-dried at 105 °C for 24 h and crushed to particles with sizes <5 mm. The obtained clay powder was mixed with different amounts of dolomitic lime . The optimal amount of water  was added to the previous mixture, in order to obtain a paste that was suitable for the elaboration of earth rendering mortars. Pastes were homogenized for 15 min and stored in an hermetically sealed plastic container in a temperature-controlled room (25 °C) for 72 h to allow for ripening. Homogeneous pastes were introduced into the molds (200 × 200 × 20 mm3) of earth renders were carried out according to the German standard DIN 18947 [Linear shrinkage, apparent density, and capillary water absorption tests of prismatic samples , and it contains insoluble residues formed essentially of quartz.Chemical analysis of the dolomitic lime shows that it is composed mainly of 54.83 wt.% of calcium oxide (CaO), 37.25 wt.% of magnesium oxide (MgO), and 7.92 wt.% of silica (SiO2), calcium carbonate or calcite (CaCO3), magnesium carbonate (MgCO3), and quartz (Q).X-ray diffraction pattern of dolomitic lime  shows th−1 (Ca-O vibration) [−1 (Ca-OH elongation vibration) [−1 (OH elongation vibration) [−1 corresponds to the vibration bands of the Mg-O bonds of magnesium carbonate [The IR spectrum of dolomitic lime is shown in bration)  and portbration)  and 3687bration) ,11. The arbonate .The DSC-TGA curves for dolomitic lime are given in The endothermic peak around 734 °C, associated with an important mass loss of 10.8 wt.%, is related to the decarbonation of calcite , followi2).Scanning electron microscopy (SEM) of dolomitic lime a shows aX-ray diffraction and differential scanning calorimetry (DSC) are the two techniques used to explore the mineralogical characteristics of the amended earth renders with dolomitic lime. The different tests were performed on the powders (ϕ < 80 μm) of each grade after 90 days of curing.X-ray diffraction was performed on the sample powders of the earth renders reinforced with dolomitic lime, in order to identify new formed crystalline phases. The diffractograms of these samples are presented in The DSC curves of the earth renders samples  highligh2 and Mg(OH)2 obtained by the hydration of the dolomitic lime. The basic character of such a medium favors this pozzolanic reaction, as shown by the pH increase of the clay–dolomitic lime mixtures  and (6), respectively:-Solubilization in a basic medium of fine ground quartz or silica from silicates of the kaolinite, according to chemical Equation (7):-Reaction in aqueous medium of the formed calcium ions Ca2+and Mg2+ with solubilized silica (H2SiO42−) to form CSH and MSH, according to chemical Equations (8) and (9):To elucidate the formation of CSH and MSH, the evolution of the intensities of the basal peak of kaolinite (2θ = 12.36°) and main peak of quartz (2θ = 26.6°), as a function of dolomitic lime amount , were himixtures . AdditioThe linear shrinkage, apparent density, and accessible porosity of earth renders have been evaluated. The obtained results of these physical properties are reported in The linear shrinkage of the earth renders samples  decreaseThe results in The hydric behavior of earth renders is a very important criterion for their acceptability in the rehabilitation of buildings. For this purpose, the capillary water absorption and spray tests have been carried out on the manufactured earth renders.The evolution of the water absorption coefficient by the capillarity of earth renders with dolomitic lime additions is presented in Under the effect of rains, earth renders samples can undergo more or less pronounced disintegration, depending on their quality. The impact of rains on different earth renders was evaluated by the spray test, using artificial rain. The evolution of the mass loss of the earth renders samples, as a function of the dolomitic lime content after the erosion test, is presented in Burkina Faso is a developing country and, in particular, a Sahel country, subject to high temperatures. It is confronted with the major problem of significant savings, due to energy consumption related to the use of air conditioning and ventilation systems. For this purpose, thermal conductivity was carried out with earth renders samples, in order to evaluate their thermal conductivity. The aim of the development of earth renders will be to offer a good thermal comfort to the buildings, while decreasing the expenses in electrical energies, which are expensive for the majority of the Burkina Faso population. The results of the thermal conductivity (λ) of the earth renders samples with dolomitic lime additions are presented in 2), according to the German standard [Building materials are subjected to stresses and react to simple abrasion, compression, and bending stresses. The primary quality of a building material is to resist to these effects, without being altered. The mechanical resistance is, therefore, an important parameter for the selection of building materials. standard . The resstandard , which sThe flexural and compressive strengths of earth renders with dolomitic lime additions are presented in The main objective of this work was to manufacture resistant, durable (water resistant), and thermally-insulating earth renders. For this purpose, the clayey raw material KOD and dolomitic lime were characterized, in order to use them for the elaboration of earth renders. The results of the mineralogical and chemical characterization showed that the clay KOD was composed mainly of kaolinite (62 wt.%), quartz (31 wt.%), and goethite (2 wt.%), and the dolomitic lime was composed mainly of calcite, portlandite, and magnesium carbonate. The addition of dolomitic lime to the clayey soil induced the formation of calcium silicate (CSH) and magnesium silicate (MSH) hydrates. These CSH and MSH were mainly formed from the pozzolanic reaction between finely ground quartz and weak silica from kaolinite in basic medium. These formed hydrates improved the physical, hydric, thermal, and mechanical properties of the earth renders. This improvement was due to the formation of CSH and MSH, which play the role of binders, allowing for the cohesion of the isolated particles of the clayey matrix, making the composite materials more compact. The earth renders are characterized by good mechanical strength and water resistance, as well as low thermal conductivity; therefore, they are suitable for habitats in dried climates, such as Burkina Faso. The hygrothermal properties, initiated in this study by the measurement of thermal conductivity, will have to be deepened, in particular, by taking the water vapour exchanges with these earth renders into account. Tests such as the measurement of water vapour sorption–desorption and moisture buffer value (MBV) would complete the hygrothermal characterization and make it possible to show the interest of using these earth renders reinforced with dolomitic lime to improve the comfort of habitats.


Pteropus medius bat roosts identified near the locations of human Nipah cases in Bangladesh during 2012–2019. Pooled bat urine was collected from 23 roosts; 7 roosts (30%) had >1 sample in which Nipah RNA was detected from the first visit. In subsequent visits to these 7 roosts, RNA was detected in bat urine up to 52 days after the presumed exposure of the human case-patient, although the probability of detection declined rapidly with time. These results suggest that rapidly deployed investigations of Nipah virus shedding from bat roosts near human cases could increase the success of viral sequencing compared with background surveillance and could enhance understanding of Nipah virus ecology and evolution.Knowledge of the dynamics and genetic diversity of Nipah virus circulating in bats and at the human-animal interface is limited by current sampling efforts, which produce few detections of viral RNA. We report a series of investigations at  Henipavirus) that has caused outbreaks of neurologic and respiratory disease in humans and livestock in Bangladesh, India, Malaysia, Singapore, and the Philippines  is the major reservoir of Nipah virus in Bangladesh and India  at any given time  values from qRT-PCR were not reported for all tests, we used the proportion of positive aliquots as a proxy for the intensity of virus shedding in bats, assuming that roosts with higher virus concentrations in urine would produce more positive aliquots. We then analyzed changes in the proportion of positive aliquots across roosts along 2 time axes. We aligned dates to the number of days since the presumed exposure date of the first human spillover Nipah case associated with each roost site. We then aligned roost-sampling dates to the number of days since the start of the calendar year for comparison. We fit binomial linear models to estimate the probability of detecting a Nipah virus–positive aliquot at each roost along each time axis.P. medius bat roost in Faridpur District during 2007–2012 as part of a longitudinal study; from visits to different roosts throughout Bangladesh during 2006–2011 as part of a cross-sectional spatial analysis; or as part of Nipah virus outbreak investigations in 2009, 2010, and 2012. Urine samples were either collected from individual bats or from underneath roosts. For these comparisons, we considered each roost visit as a discrete sampling event, including repeat visits to the same roost. Ignoring the initial visits to 7 roosts near 5 suspected human cases that were Nipah virus–negative, the 23 roosts in our study were sampled across 47 visits. We made comparisons between studies for the number of sampling visits with positive Nipah detections and the number of positive urine samples  across all sampling visits or during the first visit to each roost. We evaluated comparisons by using a χ2 test of proportions or Fisher exact test. We considered statistical tests significant if p values were <0.05.To evaluate the utility of sampling bat roosts near human Nipah virus cases as a surveillance approach, we compared the rate of successful Nipah virus detections from this study to data reported by Epstein et al.  was 0.66 (95% CI 0.42–0.84) . This prRoost urine samples from our study and individual urine samples from longitudinally sampled roosts in Epstein et al. ; the detNipah virus spillover from bats occurs sporadically in Bangladesh, so surveillance that optimizes viral detection in bats is a challenge. In contrast with cross-sectional or longitudinal bat roost surveillance used previously . RepeateThis study also provides critical information about the timing of Nipah virus shedding in bats in Bangladesh. Longitudinal surveys have shown that Nipah virus shedding from bats is sporadic throughout the year   Table 2.P. medius across Bangladesh, but increasing the number of detections is still crucial, especially given the few Nipah virus isolates currently available (n = 11). Reactive roost investigations could be complemented with additional roost surveys outside of surveillance areas to learn more about Nipah virus transmission and genetic diversity in bat populations across Bangladesh.Our case investigations were also limited to the catchment area of 3 surveillance hospitals and the winter seasonality of Nipah virus spillover surveillance. This design systematically misses virus shedding events at bat roosts outside the surveillance area or during seasons when humans are not drinking fresh date palm sap (This study provides proof of concept that reactive investigations of bat roosts near human Nipah virus cases can complement ongoing surveillance efforts and could increase the likelihood of viral detection and sequencing. Improvements in virus detection would aid in characterizing the genetic diversity of Nipah viruses circulating in bats and identify novel genotypes that might pose pandemic threats. Furthermore, these data provide evidence that viral shedding can continue for weeks after an initial spillover event, posing a hazard for additional contamination. Precise knowledge of when bats are shedding Nipah virus could be used to deploy public health campaigns more efficiently, such as by using barriers to prevent bat access to date palm sap (Additional information about Nipah virus detection at bat roosts after spillover events, Bangladesh, 2012–2019Additional data used in study of Nipah virus detection at bat roosts after spillover events, Bangladesh, 2012–2019


New and emerging plant diseases are caused by different pathogens including viruses that often cause significant crop losses. Badnaviruses are pararetroviruses that contain a single molecule of ds DNA genome of 7 to 9 kb in size and infect a large number of economically important crops such as banana and plantains, black pepper, cacao, citrus, grapevine, pineapple, sugarcane, sweet potato, taro, and yam, causing significant yield losses. Many of the species in the genus have a restricted host range and several of them are known to infect a single crop. Combined infections of different virus species and strains offer conditions that favor the development of new strains via recombination, especially in vegetatively propagated crops. The primary spread of badnaviruses is through vegetative propagating materials while for the secondary spread, they depend on insects such as mealybugs and aphids. Disease emerges as a consequence of the interactions between host and pathogens under favorable environmental conditions. The viral genome of the pararetroviruses is known to be integrated into the chromosome of the host and a few plants with integrants when subjected to different kinds of abiotic stress will give rise to episomal forms of the virus and cause disease. Attempts have been made to develop management strategies for badnaviruses both conventionally and using precision breeding techniques such as genome editing. Until 2016 only 32 badnavirus species infecting different crops were known, but in a span of six years, this number has gone up to 68. The current review highlights the emerging disease problems and management options for badnaviruses infecting economically important crops. Caulimoviridae and contain 68 species demarcated based on the sequence identity in the conserved reverse transcriptase (RT)/ribonuclease H (RNase H) coding region [Caulimoviridae consists of 11 genera including Badnavirus, namely, Caulimovirus (12 species), Cavemovirus (three species), Dioscovirus (one species), Petuvirus (one species), Rosadnavirus (one species), Ruflodivirus (one species), Solendovirus (two species), Soymovirus (one species), Tungrovirus (one species), and Vaccinivirus (one species) [Dioscovirus and Ruflodivirus have no particle morphology while badnaviruses and tungrovirus have bacilliform particles and the rest of the viruses have isometric particles. Genomes of all pararetroviruses comprise a double-stranded DNA and they replicate through an RNA intermediate. However, in contrast to retroviruses, integration of the viral genome is not mandatory for the replication of pararetroviruses. Instead, they accumulate as minichromosomes in the host nucleus. Illegitimate and generally fragmented integration occurs once in every million years [Badnaviruses are plant pararetroviruses that belong to the family g region ,2. The fon years . Particles of badnaviruses are approximately 30 nm × 120–150 nm in size and their genome consists of a circular, double-stranded DNA genome of 7.2–9.2 kb coding for a minimum of three open reading frames (ORFs) ,5. RepliBadnaviruses are one of the emerging groups of viruses infecting a large number of economically important crop plants, especially in the tropical and subtropical regions of the world . The numAlthough badnaviruses occur throughout the world both in monocots and dicots, they cause economically important diseases in tropical and subtropical regions on perennial crops such as banana, black pepper, citrus, cocoa, grapevine, pineapple, sugarcane, sweet potato, taro, and yam. Many of the species in the genus have a restricted host range and several species are known to infect a single crop   Figure . This kiThe viral genome of a few badnaviruses and other pararetroviruses is known to be integrated into the chromosome of the host through illegitimate recombination. These sequences are known as endogenous pararetroviruses (EPRVs) ,12,13,14Badnaviruses cause variable symptoms ranging from asymptomatic, mild, and moderate to severe in the hosts that are influenced by abiotic factors, especially environmental conditions and the nutrient status of the crop . Hence, Musa accuminata and M. balbisiana express severe symptoms of streak upon tissue culturing and they assumed to be due to the expression of virus from the endogenous sequence.Banana streak virus disease was first reported in Morocco in the year 1974 by the Lockhart group from the University of Minnesota. Later it was reported to occur across the world including many countries in Africa, Asia, Central, and South America, and the Pacific. It is now known that wherever bananas are grown the streak virus also exists. Banana streak virus induces typical chlorotic or necrotic streak symptoms not only on the leaf lamina but also on the midrib, leaf sheaths, and peduncle. The chlorotic streaks are narrow, small, discontinuous, and parallel to the veins and subsequently, these streaks are turned into necrotic, sometimes splitting of the pseudostem is observed to be due to the virus infection. Internal necrosis of the pseudostem, abnormal bunch emergence by piercing through the pseudostem, and necrotic streaks on fruits have also been recorded. Peel splitting and necrotic spots on fruits are reported to occur. The symptoms are not uniformly distributed on all leaves of the infected tree, and the temperature plays a major role in the type and severity of the of symptoms. The virus titer and the symptoms keep changing due to the temperature and often there will be symptomless growth for a long period, and it reappears suddenly on a new leaf. At 22 °C plants tend to express the symptoms of streak conspicuously whereas above 28 °C to 35 °C, the symptoms are invisible and if the plants are moved to a growth chamber at 22 °C the symptoms are significantly increased and become severe ,19. PlanPlanococcus citri, which transfers the virus in a semi-persistent manner. Lockhart et al. [BSVs are primarily transmitted through infected planting materials such as corms, corm bits, and infected plants mass propagated through tissue culture and the secondary transmission is through sap-feeding mealy bugs such as t et al.  reportedt et al. ,23 report et al. ,25 descrt et al.  reportedt et al.  reportedMusa phylogeny [In bananas, being a clonally propagated fruit crop, detection of viruses in the suckers or corms or corm bits is essential to ensure virus-free planting materials to the growers. Sensitive detection methods are the key to certifying the germplasm and mother plants used for mass propagation free of viruses. BSVs are serologically heterogeneous, and hence serological-based detection is cumbersome; however, IC-RT-PCR is applied using antiserum raised against a mixture of BSVs and SCMV . Moreovehylogeny ,29. Recohylogeny . The breA real-time PCR was reported for the rapid detection of episomal BSOLV and BSMYV , immunocElimination of virus from the infected propagule using cryotherapy has become successful for many plant viruses. Helliot et al.  reportedPiper nigrum) caused by the badnavirus, Piper yellow mottle virus (PYMoV) is reported in Brazil, China, India, Indonesia, Malaysia, Philippines, Sri Lanka, Thailand, and Vietnam [Piper yellow mottle virus (PYMoV) based on bacilliform virus particles, transmission by mealybug, its weak serological relationship with other badnaviruses, and its partial nucleotide sequence of the reverse transcriptase (RT)/RNase H region of the virus. Later association of PYMoV with black pepper was reported in Brazil [Piper species such as P. betle (betelvine), P. longum (Indian long pepper), and many other wild Piper species [Ferrisia virgata and Planococcus citri) and black pepper lace bug [The yellow mottle disease  of black pepper  was first discovered in Ghana in 1936 and later in other West African countries such as Ivory Coast, Liberia, Nigeria, Senegal, Sierra Leone, and Togo [Planococcoides njalensis and Planococus citri and at least other 12 species of mealybugs and also through seeds and grafting [Cacao swollen shoot disease (CSSD) of cacao  were reported [Initially, the disease was thought to be caused by different strains of the virus that cause mild, moderate, and severe symptoms. Later, viral genomic sequence data indicated that many distinct badnavirus species are associated with the disease. Currently, ICTV lists ten different cacao swollen shoot virus species associated with the disease in different cacao-growing regions. They include cacao bacilliform Sri Lanka virus (CBSLV), cacao mild mosaic virus (CaMMV), cacao swollen shoot CD virus (CSSCDV), cacao swollen shoot CE virus (CSSCEV), cacao swollen shoot Ghana M virus (CSSGMV), cacao swollen shoot Ghana N virus (CSSGNV), cacao swollen shoot Ghana Q virus (CSSGQV), cacao swollen shoot Togo A virus (CSSTAV), cacao swollen shoot Togo B virus CSSTBV, and cacao yellow vein banding virus (CYVBV) ,86. The reported . Mixed ireported . Recombireported .Serological and PCR amplification assays, though developed, failed to detect the virus in all symptomatic plants owing to the association of many badnaviral species in the disease. In a study using 189 samples, the detection potential of the primers varied from 4% to 23% with the four best primers  indicatiDioscorea bacilliform virus (DBV) infects several species of yam and negatively impacts production in all the regions of the world where the crop is grown. The common symptoms are chlorosis on veins and necrosis, and leaf distortions such as crinkling and puckering have been noticed. DBVs are primarily spread through vegetatively propagated tubers of yam which helps the virus to perpetuate, and it is also transmitted mechanically through the sap; secondarily, they are transmitted by multiple species of mealybugs of the family Pseudococcidae. Turaki et al.  reportedDioscorea alata. The complete genome sequence of two isolates of DBALV had 7413 and 7415 nt with three potential ORFs coding 29, 25, and 228 KDa proteins. Subsequently, two more isolates from Benin with 7262 and 7276 nt that showed 62% similarity with DBALV were named Dioscorea sansibarensis bacilliform virus (DBSNV). Later, another six distinct DBV genomes classified to be different species were completely sequenced; the six DBVs are, viz., dioscorea bacilliform AL virus 2 (DBALV2), dioscorea bacilliform ES virus (DBESV), dioscorea bacilliform RT virus 1 (DBRTV1), dioscorea bacilliform RT virus 2 (DBRTV2), dioscorea bacilliform RT virus 3 (DBRTV3) and dioscorea bacilliform TR virus (DBTRV) [Both DBVs and eDBVs have been characterized by genome sequencing. The first complete genome sequence of DBV named DBALV is from Nigeria and infects  (DBTRV) ,102,103. (DBTRV)   .DBV genomes are known to be integrated into the genomes of yam, which complicates the detection of the virus ,105. HigAphis illinoisensis [Planococcus ficus) [Grapevine is infected by three badnaviruses of which the grapevine vein-clearing virus (GVCV) is one of the most important emerging viruses whose distribution is, so far, limited to the USA. GVCV causes symptoms such as vein-clearing of leaves, reduced internode length, and vine decline syndrome and it is transmissible by the aphid oisensis ,108. Graoisensis ,113,114,oisensis . Besidess ficus) ,116. TheAnanas metavirus (AMtV) [Dysmicoccus neobrevipes, D. neobrevipes, and Planococcus citri) [Virions of pineapple bacilliform virus (PBV) were first detected in pineapples , and lats (AMtV) . Hawaiias (AMtV) . The RT s (AMtV) , whereass (AMtV) ,121. Amos citri) ,119,120.(SCBV) was from Cuba in 1985 followed by many other sugarcane-growing countries of the world through germplasm exchange due to lack of characteristic symptoms and vegetative propagation of the sugarcane [Sacharicoccus sachhari and Dysmicoccus boninsis [Chenopdoium quinoa, and tobacco [S. robustum, S. spontaneum, S. barberi, Erianthus arundinaceus, and E. ravennae [The first recorded report of sugarcane bacilliform virus ugarcane ,123. Thougarcane . It is sboninsis ,126,127.boninsis . Even thboninsis . Unlike ravennae .Saccharum hybrids from India. Recently the occurrence of SCBV and its variability was reported in Ethiopia [Saccharum inter-specific hybrids grown in China.Currently, ICTV recognizes four species, namely, sugarcane bacilliform Guadeloupe A virus (SCBGAV), sugarcane bacilliform Guadeloupe D virus (SCBGDV), sugarcane bacilliform IM virus (SCBIMV), and sugarcane bacilliform MO virus (SCBMOV) while other isolates are still considered only as tentative species. Of these, the SCBMOV was the first to be completely sequenced, followed by SCBIMV ,130. TheEthiopia . High geEthiopia ,136. TheEthiopia . The intEthiopia  reportedEthiopia  reportedThe variable region from SCBV was shown to serve as a promoter for high-level expression of foreign genes in both monocot and dicot transgenic plants ,140. miRSweet potato pakakuy virus (SPPV) is a collective name coined for two distinct badnaviruses, namely, sweet potato badnavirus a (SPVa) and sweet potato badnavirus b (SPVb) obtained through siRNA sequencing of a virus-infected sweet potato landrace from Peru . The comColocasia esculenta) is a perennial aroid crop grown for its edible corms in Southeast Asia and the Pacific Island countries is infected by two badnaviruses, namely, taro bacilliform virus (TaBV) and taro bacilliform CH virus (TaBCHV) [Pseudococcus solomonensis) [Taro ((TaBCHV) . The disonensis) ,148. TaBonensis) . TaBCHV onensis) . Currentonensis) . Yang etonensis)  reportedonensis) ,153, besZamia fischeri [Smallanthus sonchifolius while commelina yellow mottle virus, the type species of the genus, Badnavirus, infects the weed plant (Commelina diffusa) [Badnaviruses are also reported to infect several ornamentals, medicinal herbs, fruits, trees, and weeds ,188,189.fischeri . Virusesfischeri ,184,185,fischeri ,187. Thediffusa) . The majp-value lower than a Bonferroni-corrected cut-off of 0.05. Only recombination events detected by five or more of the different methods were considered to yield a statistically reliable prediction. Recombination events were considered credible when an event was identified by at least three detection methods with an associated p value < 0.05. The present analysis detected a total of 34 independent recombination events, the majority of which are located between the intergenic region and ORF 2  v.4 with default settings . The stand ORF 2 . Of thesnd ORF 2 . Among and ORF 2 . For recBadnaviruses are emerging viruses that cause significant economic loss in yield and quality, especially in the tropical region of the world in crops such as banana, black pepper, citrus, cacao, grapevine, sugarcane, taro, and yam. ICTV listed 68 species in the genus with many hosts  infected by more than one species. In general, viruses in the genus have a restricted host range, infecting a limited number of hosts. The majority of the badnaviruses are transmitted by different species of mealybugs in a semipersistent manner. Wild and uncultivated plants infected with badnaviruses usually remain symptomless or show only mild symptoms. The severity of symptoms of badnavirus-infected cultivated plants generally depends on abiotic factors such as temperature, relative humidity, light intensity, and nutrition of the plants. Hence, more studies are needed on the emerging problems of badnaviruses in the light of changing climatic conditions to identify the factors that trigger symptom development and the severity of the diseases in different hosts. A few studies have also indicated the role of soil pH and nutrients, including micronutrients in symptom development, also need detailed investigation. Similarly, the effect of beneficial and pathogenic organisms including fungal, bacterial, and viral on the badnavirus-infected plants also need to be studied. The information generated from the abovementioned studies would help to develop a strategy to manage the disease.Currently, the complete genome of many badnaviruses has been determined and many useful assays based on PCR, LAMP, and RPA combined with the lateral flow have been developed for the quick and sensitive detection of viruses that help in the production of virus-free plants. Similarly, methods such as RCA, in situ hybridization, and RT-PCR and IC-PCR are available to differentiate plants infected with episomal and endogenous badnaviruses. The recombinant analysis predicted the occurrence of recombinants among badnaviruses and also suggested the existence of hitherto undetermined diversity in badnaviruses. The intergenic regions of a few badnaviruses have proven that they can be used as promoters for the expression of foreign genes in different hosts. However, the role of different genomic components of the virus on disease development is still unknown.Methods such as thermo- and chemo-therapies combined with meristem tip culture were reported for the production of virus-free plants of a few badnaviruses. This should be expanded to more hosts, especially the germplasm accessions that are used to breed crops resistant to different pathogens. So far, no varieties resistant to badnaviruses are reported. Hence, more research efforts are needed to develop varieties resistant to badnaviruses using both conventional and biotechnological including gene editing approaches. Recently, endogenous badnaviral sequence-edited banana plants lost their ability to give rise to the episomal form. The role of integrated badnavirus sequences that might impart immunity to their hosts requires further study.


In vitro experiments showed that the antiviral activity of the Ren's oligopeptide mainly targets the replication process after virus enters the cell. Therefore, Ren's oligopeptide is a promising drug against influenza and COVID-19.Influenza virus and SARS-CoV-2 virus are two important viruses that cause respiratory tract diseases. The high-frequency mutation of the two types of viruses leads to failure of the durable immune protection of vaccines, meanwhile it also poses continuous challenges to the development of antiviral drugs. Traditional Chinese medicine contains large number of biologically active compounds, and some of them contain broad-spectrum antiviral ingredients. In this study, we extracted antiviral active ingredients from medicinal and edible plants by biotransformation and enzymatic hydrolysis as a drug, and we named this drug Ren's oligopeptide. Further, we analyzed the antiviral activity of this drug and found that Ren's oligopeptide could inhibit the replication of influenza virus and SARS-CoV-2 virus with high anti-virus activities.  Influenza is highly contagious, which cause huge economic losses. Therefore, influenza is listed as one of the major infectious diseases that seriously threaten human health . CurrentThe new coronavirus pneumonia caused by SARS-CoV-2 virus poses a huge challenge to global public health, causing 560 million infections and 8.67 million deaths worldwide by July 2022 . Currentin vitro. Therefore, it can be used as a candidate drug for the treatment of respiratory tract infections caused by influenza A virus and SARS-CoV-2 virus.Influenza virus and SARS-CoV-2 virus are two important pathogen causing respiratory tract infection, both viruses are highly contagious with high rates of mutation, which makes clinical antiviral treatment difficult . ChineseRen's oligopeptide is prepared from six plant ingredients through enzymatic fermentation. Specifically, the fermented raw materials were chopped and mixed at the proportions of 20% ginger, 20% garlic, 20% onion, 10% banana peel, 20% bitter melon, and 10% deionized water. The fermentation enzyme (Amylase enzyme) was extracted from the Geotrichum candidum strain, and the concentration was measured by the QuantiPro ™ BCA kit . Amylase enzyme was added to the mixture at a weight ratio of 1:10,000, followed by 8-day aseptic fermentation at 25°C and the precipitate removal. The concentration of flavonoid anthocyanin in the products was determined to measure the degree of fermentation. Afterwards, the supernatant was sterilized and stored in aliquots at 4°C. The color of Ren's oligopeptide solution is brown and the concentration of Ren's oligopeptide was 900 μg/ml.2 for 48 h. The influenza and SARS-CoV-2 virus strain used in this study were H1N1-PR8 and nCOV2019BetaCoV/Wuhan/WIV04/2019.MDCK (ATCC CCL-34) and Vero-E6 (ATCC CCL-81) cells were passaged in DMEM medium  containing 10% FBS  and 1% penicillin-streptomycin . When reached 95% confluency, the cells were digested with 0.25% trypsin  and transferred into flasks, then incubation at 37°C and 5% CO4 cells per well and incubated at 37°C and 5% CO2 for 24 h. Ren's oligopeptide was two-fold serially diluted in PBS, then added to the 96-well plate with 6 replicates per group. In addition, a drug-free group treated with PBS was used as control group. Cell viability was detected with Cell Counting Kit 8  after 48 h incubation. The 10 μl/well of WST-8 solution was added into each well, and then incubate protected from dark at 37°C for 2 h. The absorbance at 450 nm was measured by a microplate reader.MDCK and Vero-E6 cells were separately seeded into 96-well plates at 1 × 10To investigate the antiviral effect of Ren's oligopeptide, TCID50 was performed. The experiment grouping was as follows: (1) control group: cells without virus infection; (2) virus infection group: cells were incubated with 100 TCID50 viruses; (3) virus pretreatment group: virus and Ren's oligopeptide were mixed at room temperature for 30 min before infection; (4) cell pretreatment group: cells were incubated with Ren's oligopeptide for 24 h and then infection with 100 TCID50 virus; (5) virus adsorption-blocking group: cells were treated with Ren's oligopeptide for 1 h during the virus adsorption period; (6) virus replication-blocking group: the diluted Ren's oligopeptide was added after virus adsorption.4 of MDCK or Vero-E6 cells per well were seeded into a 96-well plate, and cultured at 37°C and 5% CO2 for 24 h. The diluted drug (1:16) was added to the pretreatment group. An equal volume of PBS was added to other groups. After 24 h incubation, each group was infected with the virus with a dose of 100 TCID50. The supernatants were collected after 48 h incubation for RNA extraction, and the virus copy number was detected by qRT-PCR.The antiviral effect of Ren's oligopeptide was analyzed at its maximum non-toxic concentration. 1 × 10To test the dose-dependent antiviral effect of Ren's oligopeptide, the drug was two-fold serially diluted with its maximum non-toxic concentration as initial concentration. After infection for 1 h, the diluted drug was added to the virus replication-blocking group. After 48 h cell culture, the supernatant was collected for RNA extraction. The experiments were performed in triplicates.For SARS-CoV-2 virus, the cell supernatants were centrifuged at 3,000 rpm for 5 min, then virus lysate was added, followed by virus inactivation according to Wuhan National Biosafety Laboratory BSL-3 SOP. Viral RNA was extracted with Takara viral RNA extraction kit , and the viral copy number was detected by qRT-PCR using the Luna Universal One-Step RT-qPCR Kit . Plasmid pUC57-RBD contains receptor-binding domain (RBD) sequence was used to generate a standard curve. The primers pair for SARS-CoV-2 detection were RBD-F (CAATGGTTTAACAGGCACAGG) and RBD-R (CTCAAGTGTCTGTGGATCACG), and the detection sequence is 5'6FAM-ACAGCATCAGTAGTGTCAGCAATGTCTC-3'BHQ1. The H1N1 influenza viral genomic RNA was extracted using a nucleic acid extractor, and the viral copy number was detected by qRT-PCR . Standard curves were created using plasmid pUC57-NP harboring the nucleoprotein (NP) sequence. The primers pair for H1N1 virus detection were NP-qF (GAGCCGGAACCCAGGGAATGCTGA) and NP-qR (CAAAGTCGTACCCACTGGCTACGGCAG), and the detection sequence is 5'6FAM-TCCATACACACAGGCAGGCAGGCAGGAC-3'BHQ1. All the experiments were performed in triplicates.The virus inhibition rate was calculated according to the following formula:t-test was conducted to assess the statistical significance. P < 0.05 was considered statistically significant.Virus inhibition rate =  × 100%. CC50 (50% cytotoxicity concentration) and EC50 (50% effective inhibition concentration) were calculated by non-linear regression according to cell viability and viral inhibition rate. The P = 0.34). However, the cell viability was significantly decreased in the 113 μg/ml treatment group  (P = 0.64), and the cell viability was significantly lower in drug treatment group than in the control group when the drug concentration was 113 μg/ml, (<20.33%)  . Similar<20.33%) . Finally<20.33%) . Since tP = 0.71), indicating that the drug had no direct killing effect on the virions. Compared with that in the control group, the virus copy number in the cell pretreatment group was decreased with an inhibition rate of 28.34%, but there was no significant difference (P = 0.085). The inhibition rate in the virus adsorption blocking group was 13.19% with no significant difference from the control group (P = 0.394). When the drug was added to cells during viral replication, the replication of the virus was significantly reduced with an inhibition rate of 99.74% (P = 5.1 × 10−5)  . OverallFurther, we analyzed whether the antiviral effect of Ren's oligopeptide was dose-dependent by serial dilution. Our results showed that with the decreasing drug concentration, the virus copy number was increased , and theP = 0.025). The virus copy number was 7.27% lower in the cell pretreatment group than in the control group, but it was not statistically significant (P > 0.99). When it was added to the Vero-E6 cells during the virus adsorption stage, the drug did not show any inhibitory effect. However, when the drug was added 1 h after virus infection, the virus replication was significantly lower than that in the control group (P = 1.56 × 10−5) with an inhibition rate of 56.34% (We assessed the anti-SARS-CoV-2 virus effect of Ren's oligopeptide against SARS-CoV-2 using Vero-E6 cells. The results showed that the virus copy number in virus pretreatment group displayed a downward trend with an inhibition rate of 12.45% (f 56.34% . Similarf 56.34% , D. Therin vitro. Ren's oligopeptide showed a significant antiviral effect on the influenza virus H1N1 at the maximum non-toxic concentration with an anti-H1N1 inhibition rate of up to 99%. The direct incubation of the drug with the virus did not inhibit the replication of the H1N1 virus, indicating that the drug could not directly kill the virus to exert its antiviral effect. The addition of the drug during virus adsorption stage only resulted in a limited antiviral effect. In both the H1N1 infection group and the SARS-CoV-2 infection group the addition of drug during the virus replication stage exhibited the optimal inhibitory effect on the virus replication, indicating that the drug mainly played a role by targeting the virus replication stage.Ren's oligopeptide is fermented from edible plant ingredients with a wide range of raw materials and low side effects. In this study, we confirmed Ren's oligopeptide antiviral activity against SARS-CoV-2 virus and influenza virus The antiviral effect of Ren's oligopeptides results from the synergistic action of various active ingredients in the fermentation materials. Garlic is known as a natural antibiotic with broad-spectrum antiviral effects . Organosin vivo antiviral effect of this drug remain to be further investigated.Based on the above findings, we proposed that Ren's oligopeptide can be used as a candidate drug for the treatment of respiratory diseases caused by influenza virus and SARS-CoV-2 virus infection. The limitation of this study is that the detail antiviral mechanism and in vitro antiviral analysis, we determined that the maximum non-toxic concentration of Ren's oligopeptide is 56 μg/ml, and Ren's oligopeptide has a significant antiviral effect on influenza virus and SARS-CoV-2 virus, and the antiviral activity is mainly manifested in the replication phase after the virus enters the cell.Ren's oligopeptide is fermented from edible plants with antiviral effects, it contains a variety of antiviral active compounds. Through the The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.CY: conceptualization. CY and YH: methodology and formal analysis. XR: software and investigation. YH and XR: validation. LS: resources and writing and editing. LS and CY: data curation and project administration. All authors contributed to the article and approved the submitted version.


Congenital cytomegalovirus (cCMV) infection can cause severe neurological damage, growth retardation, hearing loss, and microcephaly in infants. We aimed at assessing healthcare costs of infants with recorded cCMV diagnosis in an administrative claims database in the first 2 years of life.90 cohort) to infants without cCMV diagnosis (“representative” controls) and infants with cCMV diagnoses ≤ 21 days after birth plus specific symptoms (cCMV21-S) to infants without cCMV and any ICD-10-GM records (besides Z00-Z99) until 4th preventive health check-up . Due to missing data, mean imputation was applied for aids and remedies costs.We conducted a retrospective, controlled cohort study using German claims data from the Institute for Applied Health Research Berlin (InGef) database. Incremental healthcare costs during the first and second year of life were assessed by matching (1:60) infants with cCMV diagnoses ≤ 90 days after birth  healthcare costs were significantly higher in cCMV90 cases vs. “representative” controls  vs. €3091 (€863), p < 0.001), with 87.2% inpatient costs. Healthcare costs for cCMV21-S cases compared to “healthy” controls were €34,498  vs. €680 (€569), p < 0.001. Differences decreased for both comparisons in the second year but remained statistically significant.We identified 54 and 24 infants born 2014–2018 for the cCMVcCMV comprises a considerable economic burden for the German healthcare system . Attempts should be made to reduce this burden.The online version contains supplementary material available at 10.1186/s12962-022-00411-x. Herpesviridae, establishing lifelong persistence after primary infection. Seroprevalence in the general population is estimated to be 45–100% [Cytomegalovirus (CMV) belongs to  45–100% , 3. Cong 45–100% , 4. CMV- 45–100% , 6 and 0 45–100% . About 1 45–100% , 8, 40–5 45–100% .For Germany, published data on the health economic burden of cCMV in infants are limited. A cost-of-illness study using data from 2008  estimateThe present study covers one part of a large retrospective study that assessed the burden of cCMV in terms of cCMV sequelae, healthcare resource utilization and healthcare costs in infants during the first and second year of life from the perspective of the German Statutory Health Insurance (SHI). In this paper we present incremental healthcare costs of infants with cCMV compared to infants without cCMV. Due to challenges in retrospective identification of infants with cCMV , and speWe conducted a retrospective, controlled matched cohort analysis using German SHI claims data from 2014 to 2019 to assess the incremental healthcare costs of cCMV in infants with and without recorded cCMV diagnosis during their first and second year of life. The first and second year of life were defined as the first 1–365 and 366–730 days of life, respectively.We utilized the Institute for Applied Health Research Berlin (InGef) database with SHI claims data from about 60 different health insurances covering approximately 8 million lives with a well-distributed geographic representation of the German population and good external validity in terms of morbidity, mortality, and drug use .Claims data from the participating SHIs are joined in a specialized trust center, anonymized, and transferred to InGef before the data are made available for research. By German legislation, the analysis of claims data from the SHI is permitted and does not require the approval of an ethics committee.Infants in the InGef database born 2014–2018 were included in this study. Newborns needed to be continuously observable for at least 365 or 730 days of life, except for infants who deceased. Infants with pre-specified diagnoses of immunocompromising diseases , solid organ transplant, or stem cell transplant) during the first 365 days of life were excluded from the study population : all infants with a documented diagnosis for cCMV (ICD-10-GM P35.1) during the first 90 days of life irrespective of documented clinical symptoms.cCMV-cohort 1 (cCMV21-S): all infants with documented inpatient diagnosis for cCMV (ICD-10-GM P35.1) and at least one cCMV-specific symptom recorded during any hospital admission in the first 21 days of life (including birth).cCMV-cohort 2 (cCMVStudy cohortsControl group 1 (“representative”): all infants without a documented diagnosis for cCMV (ICD-10-GM P35.1) and CMV (ICD-10-GM B25) at any time during their individual observation period in the database.Control group 2 : all infants from control group 1 with no ICD-10-GM records (except Z00–Z99 codes) in the quarters of preventive check-ups for children (until 4th preventive check-up).Control groupsTwo cCMV cohorts and control groups were defined:A more detailed description of selection criteria including information on the German claims data coding system can be found in Additional file 21-S cohort included infants with recorded inpatient cCMV diagnosis during the first 21 days of life in combination with at least one pre-defined cCMV-associated symptom/disease [90 cohort included infants with cCMV diagnoses during the first 90 days of life irrespective of symptom records, as during these 90 days, retrospective diagnosis of cCMV infection can be performed by polymerase chain reaction using dried blood spots . Due to German data protection regulations, Guthrie cards must be destroyed after 3 months, making a reliable diagnosis of cCMV infection nearly impossible thereafter [A distinction should be made between congenital and postnatal CMV infection as the long-term complications and treatment options differ considerably . CMV inf/disease , 17, to ereafter .90 cohort to ”representative” controls and the comparison of the cCMV21-S cohort to a more artificial cohort of ”healthy” controls. The first comparison will produce the lowest and the latter comparison the highest increment estimate for healthcare costs concerning all four possible comparisons, providing a plausible span for cCMV-related healthcare costs of infants with recorded cCMV diagnosis from the perspective of the SHI in Germany. The two complementary comparisons  are reported in Additional file Here, we present the comparison of the cCMVHealthcare costs were analyzed as costs for outpatient care, inpatient care, outpatient pharmaceuticals, remedies, devices and aids, and summarized as total costs. Outcomes were analyzed separately for the first and second year of life.We performed a 1:60 direct matching. The matching ratio selected was data-driven based on the highest number of matches available for all group comparisons. Matching was based on gender, year-specific quarter of birth, and observability of the newborns.Descriptive analyses were performed for patient demographics, and healthcare costs in terms of mean values, medians, ranges, and standard deviations for continuous variables and absolute and relative frequencies for categorical variables.As data for remedies, devices and aids are not completely available for all individuals in the database due to technical issues with data transfers, single imputation was applied using the mean costs of infants with available data.Statistical significance tests of descriptive differences comprised non-parametric Mantel–Haenszel matched-pairs analysis for dichotomous variables and Wilcoxon rank-sum tests for continuous variables. P < 0.05 was considered as statistically significant. Additionally, for continuous variables, mean differences with 95% confidence intervals (CI) were calculated.To assess the effect of potential cost outliers, the cost analysis was repeated using 95% one-sided upper winsorization, i.e., infants with cost values above 95% percentile were not excluded, but their costs were replaced with their group’s 95% percentile cost value.90 definition. Of those, N = 34 were observable in the second year of life. In the cCMV21-S cohort, this applied to N = 24  and N = 15 infants, respectively, , whereas > 95% in both control groups showed regular birth weight  vs. €3091 (€863), p < 0.001) with a mean difference of €19,646 . For the cCMV21-S cohort, mean (median) total costs per infant were about 51 times higher compared to “healthy” controls  vs. €680 (€569), p < 0.001) resulting in a mean difference of €33,818 per infant  during the first year of life. Costs for inpatient care accounted for 87.2% (cCMV90) and 89.0% (cCMV21-S) of total mean costs and were thus the main cost driver during the first year of life in both cCMV cohorts  vs. €1132 (€562), p < 0.001) with mean difference of €3195 (CI €546–€5844). For the cCMV21-S cohort, mean (median) total costs per infant were more than six times higher compared to “healthy” controls (€4714 (€1977) vs. €699 (€441), p < 0.001), leading to a mean difference of €4015 (CI € − 1091 to €9121) during the second year of life. Costs for inpatient care remained the main cost driver in the second year of life with more than 50% of total healthcare costs occurring in the inpatient setting  healthcare costs per infant were more than three times higher in cCMVThis is the first study investigating healthcare costs in infants with recorded cCMV diagnoses during their first and second year of life for Germany using SHI claims data. Our study highlights a significantly higher burden in infants with recorded cCMV diagnosis in terms of healthcare costs compared to infants without cCMV diagnosis, suggesting that on average, one infant with recorded cCMV diagnosis produced €19,646–€33,818 more costs for the SHI during their first life year compared to one infant without cCMV diagnosis. Inpatient costs were the main cost driver. The study cohort had information on 54 infants with recorded cCMV diagnoses out of 282,582 births, which amounts to a birth prevalence of 0.02%. As other sources suggest that the actual birth prevalence of cCMV in Germany may be between 0.2% and 0.5% . Therefo90 cohort with “representative” controls, we found differences of similar magnitudes. However, the US study included infants with cCMV diagnosis performed during the complete first year of life [90 cohort comprised only infants with cCMV diagnosis during the first 90 days of life. A nationwide retrospective cohort study from the Netherlands found almost two times higher mean healthcare costs per child in the first 6 years of life in 156 infants with cCMV-infection confirmed by screening of a retrospectively recruited sample of children’s of DBS (average difference: €2544) compared to a matched cCMV-negative control group [Also internationally, only limited published data assessing comparable research questions are available. A claims data analysis from the US found that costs of infants with cCMV diagnoses during the first year of life were on average seven times higher compared to matched infants without cCMV diagnosis . When co of life , whereasol group . Howeverol group , costs ool group  or proviol group . A retrool group . They al21-S cohort with “healthy” controls. These differences in magnitude of results were expected, as the cCMV21-S cohort comprised the sub-group of presumably severe cCMV cases already symptomatic at birth, whereas the cCMV90 cohort allowed for including cases where congenital infection could still reasonably be assumed despite a potentially later symptom onset. With these two definitions we aimed at building a range for the actual healthcare costs in infants with cCMV. The definitions of the two control groups analogously followed this intention. The comparison of the cCMV21-S cohort with “healthy” controls should represent the most extreme expected differences (comparing the cCMV worst case scenario with the best-case control group scenario), whereas the comparison of the cCMV90 cohort with “representative” controls may provide more conservative incremental cost estimates. Complementary comparisons of the cCMV90 cohort with “healthy” controls and the cCMV21-S cohort with “representative” controls resulted in point estimates between the extremes described above, which may be closer to a population average . Therefore, the cCMV21-S cohort might provide a higher probability of valid cCMV diagnoses but misses out on infants with symptoms detected only after the first 21 days of life.Since clinical or laboratory data is not available in the database, it is impossible to verify if the recorded cCMV diagnoses were based on virological results. Consequently, the classification of infants in the cCMV cohorts depends to some extent on the ICD coding behavior of physicians. To face these limitations, we established a detailed identification process for cCMV infants. Furthermore, reliable diagnosis of cCMV depends on direct virus detection either during the first 14 days post-partum using polymerase chain reaction diagnosis or from day 1–21 post-partum using virus culture , 16. AddIn sum, our study population presumably consisted of infants which were symptomatic in their first 21 and likely in their first 90 days of life. Assumptions on costs of all infants with cCMV, including infants with asymptomatic cCMV or misdiagnosed symptomatic infants, cannot be made in this study, as these are likely not covered by our study population. Consequently, the cost estimates cannot be extrapolated to the overall population of infants with cCMV in Germany, as this would probably overestimate the costs per infants with cCMV for the SHI. As we only assessed the first 2 years of life, costs thereafter as well as costs of infants with cCMV but a later symptom onset  were not covered. However, we may have underestimated the total costs for symptomatic infants as certain disability support measures, like special kindergartens, are incompletely represented in the InGef database and might additionally be requested more often for children after the age of 2 years. The representativeness of cost estimates from administrative databases for the overall cCMV population has also been questioned elsewhere , 24. EveAs valid data for remedies, devices, and aids were not available for all infants, mean imputation was applied for infants with invalid data. By using the mean for imputation of missing remedies, devices, and aids data, the results for this cost domain and consequently the total costs are possibly skewed as the mean could be influenced by outliers. As the main message of this study refers to mean values, we decided to use the mean imputation for reasons of consistency.th percentile as sensitivity analysis to assess the effect of potential cost outliers. Even after exclusion of high-cost diseases and winsorization, the results show a high variability of values indicating that healthcare cost in infants with cCMV infants may differ considerably  those were excluded from the study population. Additionally, cost results were winsorized at 95Another limitation of the database and the study design is that the presented data can only to some extent provide detailed information on specific treatments. For instance, pharmaceuticals dispensed in the inpatient setting  cannot be assessed separately as these costs are usually included in the compensation schemes for diagnosis related groups (DRG). However, this also reflects a major strength of this study as it shows the overall costs and resource utilization that occurred from the perspective of SHI. By comparing respective increments between infants with cCMV and infants without cCMV, we were able to identify the cost differences which are assumed to be related to cCMV.The results of this study underline the significant health economic burden in infants with recorded cCMV diagnosis for the SHI system in Germany and indicate the need for preventive steps and a comprehensive monitoring of cCMV infection and related sequelae. With on average €19,646–€33,818 higher annual costs during the first and €3195–€4015 higher annual costs during the second year of life for infants with recorded cCMV diagnosis compared to infants without cCMV, the currently measurable additional healthcare costs per infant with recorded CMV diagnosis are substantial. Even though extrapolation of the reported per case costs to the complete dark figure of infants with cCMV in Germany would likely overestimate the health economic impact of cCMV for the SHI in Germany, a considerable budget impact remains conceivable. An important question is which measures may be helpful to reduce this significant burden of cCMV in the future. As vaccines against CMV are not available, other steps for prevention of cCMV infection need to be implemented.Systematic collection of information on cCMV infection in pregnant women and newborns could not only help raise awareness of the risks of cCMV, but also provide better estimates on the actual burden of disease. Without reliable numbers of cCMV infections, which could be provided by neonatal screening, projection of the real overall health economic burden for all infants with cCMV to national level—including days parents are absent from the job, lifelong support in case of disability etc.—remains incomplete.Due to the focus on the first two life years, our results present only a small fraction of the actual health economic burden of cCMV for the SHI for infants with recorded cCMV diagnosis and do not consider any indirect or societal costs. Further research should assess societal and lifetime costs of cCMV to understand the complete picture and attempts should be made to reduce the burden of cCMV.Additional file 1: Table S1. Exclusion Criteria for Overall Study Population. Table S2. cCMV-specific Symptoms and Sequelae. Table S3. All-cause Healthcare Costsa During the First 1-365 Days of Life After Winsorization. Table S4. All-cause Healthcare Costsa During the First 366-730 Days of Life After WinsorizationAdditional file 2. Table S1. Baseline Demographic and Clinical Characteristicsa After Matching–Complementary Comparisons. Table S2. All-cause Healthcare Costsa During the First 1-365 Days of Life, Unadjusted–Complementary Comparisons. Table S3. All-cause Healthcare Costsa During the First 366-730 Days of Life, Unadjusted – Complementary Comparisons. Table S4. All-cause Healthcare Costsa During the First 1-365 Days of Life After Winsorization – Complementary Comparisons. Table S5. All-cause Healthcare Costsa During the First 366-730 Days of Life After Winsorization–Complementary Comparisons.


Pestivirus of the family Flaviviridae mainly comprises classical swine fever virus (CSFV), bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, border disease virus (BDV), and multiple new pestivirus species such as atypical porcine pestivirus (APPV), giraffe pestivirus, and antelope pestivirus. Pestiviruses cause infectious diseases, resulting in tremendous economic losses to animal husbandry. Different types of pestivirus vaccines have been developed to control and prevent these important animal diseases. In recent years, pestiviruses have shown great potential as viral vectors for developing multivalent vaccines. This review analyzes the advantages and disadvantages of various pestivirus vaccines, including live attenuated pestivirus strains, genetically engineered marker pestiviruses, and pestivirus-based multivalent vaccines. This review provides new insights into the development of novel vaccines against emerging pestiviruses, such as APPV and ovine pestivirus.The genus  Pestivirus (from the Latin pestis—plague), belonging to the family Flaviviridae, is responsible for infectious diseases in swine, cattle, sheep, goats, and other domestic and wild animals. According to the recent reclassification of the genus Pestivirus by the International Committee on Taxonomy of Viruses (ICTV), the pestivirus taxonomy resulted in the demarcation of eleven species designated pestiviruses A through K. Due to the increasing number of diverse pestiviruses, the taxonomy of the genus Pestivirus was revised in 2017 based on nucleotide or amino acid sequence distances of complete coding sequences, combined with antigenic differences, natural host ranges, and pathology. The number of pestivirus species was expanded to nineteen by identifying eight new species designated pestiviruses L through S, including atypical porcine pestivirus (APPV), giraffe pestivirus, antelope pestivirus, HoBi-like pestivirus, Bungowannah virus, and Linda virus ..® CSF Marns or E2 of CSFV are potential marker vaccines for CSF   . WhetherThe expression levels of the foreign genes in the pestiviral vectors can be affected by various factors, such as the insertion sites and expression strategy. For example, the titers of the engineered C-strain and its mutants are relatively low, which might affect the expression levels of foreign genes. The robust adaptation of the attenuated viruses to the cells and the manufacturing techniques are required to improve the viral titers .Our group has generated three C-strain-based recombinant viruses expressing the capsid (Cap) gene of PCV2 by reverse genetic manipulation . The CapIn summary, a number of vaccine strategies have been explored to combat pestivirus infections of livestock and wildlife. However, there are several limitations in the pestivirus vaccines. Firstly, the safety of engineered vaccines based on pestiviral vectors should be evaluated in the field. This is relevant for vaccination against infectious diseases and potentially exploiting virus-based vectors in vaccination strategies where individuals are sometimes immunocompromised. Secondly, maternally derived antibodies may inhibit the immune responses of LAVs. Thirdly, subunit vaccines usually cannot provide rapid-onset and complete protection. Additionally, new-type pestivirus vaccines need a long time before they can be commercially available.+/CD4+ T cells) contributes to the protection against ASFV infection [Replicons are replication-competent RNA molecules that are incapable of generating infectious progeny viruses due to the loss of one or more structural proteins. The genome sequences encoding NS3-NS5B together with the 5′ and 3′ UTRs are the minimal elements required for autonomous pestiviral RNA replication . Signifinfection ,139. ConViral vectors hold great promise for development of multivalent vaccines to counter co-infectious diseases. Compared with other viral vectors, pestivirus-based vectors have the following advantages: (1) The infectious clones of the RNA viruses are easier to be engineered than those of the DNA viruses due to their smaller genome size of 7 to 19 kb; (2) The double-stranded RNA produced by the genome replication can enhance the immunization efficacy; (3) Viral proteins are expressed with high efficacy; (4) The replication of the RNA occurs in the cytoplasm rather than in the nucleus, thus avoiding the RNA degradation; (5) No RNA can be integrated into the DNA of the host; (6) Since they have fewer viral structural proteins and cause less immune responses against the vector, the pestiviral vectors have great potential for developing safe and effective vaccines against the animal diseases.Several CSF vaccines have been developed for oral immunization of wild boars ,147,148.Collectively, various pestivirus vaccines have been developed by different attractive strategies and have shown the advantages and disadvantages in terms of the safety, efficacy involved in the rapid-onset protection, cross-protection, and DIVA potential, which will provide new insights into the development of novel vaccines against emerging pestiviruses.