Citrivirus, while the genomes of MaVLV and MaNLV were nearly completed lacking the 5′ and 3′ termini sequences. We tentatively consider MaVLV to be members of the family Virgaviridae and MaNLV to be members of the genus Narnavirus based on the results of phylogenetic trees. The infection experiments showed that CLBV-ML2 could be detected in the inoculated seedlings of both N. benthamiana and Morus alba, while MaVLV could only be detected in N. benthamiana. All of the infected seedlings did not show obvious symptoms.Viruses-mediated genome editing in plants is a powerful strategy to develop plant cultivars with important and novel agricultural traits. Mulberry alba is an important economic tree species that has been cultivated in China for more than 5000 years. So far, only a few viruses have been identified from mulberry trees, and their application potential is largely unknown. Therefore, mining more virus resources from the mulberry tree can pave the way for the establishment of useful engineering tools. In this study, eight old mulberry plants were gathered in seven geographic areas for virome analysis. Based on transcriptome analysis, we discovered three viruses associated with mulberries: Citrus leaf blotch virus isolate mulberry alba 2 (CLBV-ML2), Mulberry-associated virga-like virus (MaVLV), and Mulberry-associated narna-like virus (MaNLV). The genome of CLBV-ML2 was completely sequenced and exhibited high homology with Citriviruses, considered to be members of the genus  Resent bravirus ,4.Morus alba) belongs to the genus Morus in the family Moraceae, which is a deciduous tree that has been grown for thousands of years in China [Mulberry crinkle leaf virus (MCLV) [Paper mulberry mosaic-associated virus (PMuMaV) [Mulberry mosaic dwarf-associated virus (MMDaV) caused mosaic and dwarfing symptoms in mulberry trees [mulberry cryptic virus 1 (MuCV1) exhibited yellow vein symptoms [mulberry badnavirus 1 (MBV-1) include mosaic structure, deformation, vein clearing and necrosis on the leaves and deformation, crumbling, and scabs on the fruits [Citrus leaf blotch virus-ML (CLBV-ML) caused symptoms of chlorotic leaf spots and occasionally witches’ broom [paper mulberry leaf curl virus 1 and paper mulberry leaf curl virus 2 (PMLCV-1 and PMLCV-2) were identified in paper mulberry plants affected by a disease with leaf curl symptoms [The mulberry   and the (PMuMaV) . With thry trees , and mulsymptoms . The syme fruits . Citrus s’ broom . In addisymptoms . Howevercitrus leaf blotch virus (CLBV), a member of the genus Citrivirus. We named it Citrus leaf blotch virus-Mulberry Alba 2 (CLBV-ML2) since it is homologous to the previously reported mulberry virus Citrus leaf blotch virus-ML [Virgaviridae and Narnavirus, with the names of Mulberry-associated virga-like virus (MaVLV) and Mulberry-associated Narna-Like virus (MaNLV) proposed. RT-PCR experiments have also been conducted with specific RdRp primers of these viruses to verify their existence. Besides, the results of virus infection experiments showed that MaVLV could infect N. benthamiana, and CLBV-ML2 could be detected in the inoculated seedlings of both N. benthamiana and Morus alba without obvious symptoms.Nowadays, plenty of old mulberry trees, which have lived for hundreds and thousands of years, are mainly distributed in the middle and lower reaches of the Yellow River, southwest, northwest, and northeast of China. In this study, Transcriptome sequencing technology was applied to eight old mulberry trees’ leaves from seven provinces in order to uncover novel viruses that co-existences with their hosts. Three viruses were detected from the sequence data, one of which is virus-ML . The othThe present study examined eight different species of ancient mulberry trees from Chinese provinces . The samples were leaves of ancient mulberry trees, which were asymptomatic at the time of collection. They were stored in sterile 50 mL conical tubes at −80 °C until further analysis.https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 15 January 2022). A conserved domain search was performed using the CDD/SPARCLE tool  of NCBI [The obtained samples were sent to Majorbio  for total RNA isolation and transcriptome sequencing and analysis. Raw data were trimmed using Trimmomatic (Version 3.90)  and trin of NCBI . TBtoolshttps://www.ncbi.nlm.nih.gov/, accessed on 15 January 2022), compared with the related viral sequences using ClustalW and analyzed using the Maximum likelihood (ML) algorithm with the LG and WAG substitution model to construct phylogenetic trees in MEGA X with 1000 bootstrap replications, respectively [The amino acid sequences of the predicted RNA-dependent RNA polymerase (RdRp) regions from MaVLV and MaNLV were used for phylogenetic analyses in this study. The sequences were obtained from National Center for Biotechnology Information . The 5′ RACE CDS Primer A and the 3′ RACE CDS Primer A was used to synthesize the cDNAs, which could include specific amplification of 5′ and 3′ end sequences, respectively. The universal primer mix and the gene-specific primers . The purified PCR product was cloned into a pESI-T vector , and the M13-F/R primer was used for positive clone detection and sequenced commercially . Finally, the whole sequence was assembled with DNAMAN software (version 9.0), and observed that an additional 66 nt at the 5′ terminus and 528 nt at the 3′ terminus was identified.The full genome sequence of the candidate CLBV-ML2 was obtained by rapid amplification of cDNA ends (RACE) using a SMARTerers GSP,  were use2PO4, 118 mL 0.2 mol/L NaOH, add H2O to 1000 mL, pH 6.8), then rub the extracted sap onto the leaves of 5 N. benthamiana and 5 Morus alba. Total RNA was extracted from system leaves after 10 days. RT-PCR was used for the RdRp sequence detection from the samples’ total RNA and inoculated plants’ total RNA. Following the manufacturer’s instructions, the RT reaction was performed with Hifair® III 1st Strand cDNA Synthesis Kit  with Oligo (dT)18 primer. One microliter of cDNA was used as a template for the PCR reaction in the presence of primers of RdRp detection. The reaction procedure was 94 °C for 3 min; 94 °C for 30 s; 55 °C for 30 s; 72 °C for 1 min (30 s/kb); 72 °C for 5 min, and end of 4 °C, which produced 1295 bp, 729 bp, and 1298 bp fragments, respectively.Mechanical inoculation was used for virus infection. The basic procedure was to grind up the samples from ancient mulberry trees and add the appropriate amount of phosphate buffer  strain fly97447 (KX883767) and Broome virga-like virus 1 (BVLV1) isolate BVGL1/pool-2 (MT498833), which are unclassified virgaviruses found in invertebrates, respectively. Four ORFs were found using the ORFfinder web tool. ORF1 is positioned at 98–7372 nt in the genome, predicted to encode a 2424 aa polyprotein including a viral methyltransferase , an FtsJ-like methyltransferase , a viral RNA helicase , and an RNA-dependent RNA polymerase 2 domain . ORF2 is positioned at 8256–9257 nt in the genome, which encodes unknown proteins. ORF3 and ORF4 are positioned at 9291–10061 nt and 10081–10614 nt in the genome, predicted to encode 154 and 151 aa SP24, respectively A.CLBV-ML2 has an 8794 nt genome with 66 nt UTR at its 5′ terminus and 528 nt UTR at its 3′ terminus. The 5′-UTR with a length of 66 nt shares 98.48% nt identity with that of CLBV-PY2 (MW713063), and the 3′-UTR with a length of 528 nt shares 95.83% nt identity with CLBV-ML (MT767171). The complete genomic sequence shares 83.08% and 81.87% identity with CLBV-PY2 and CLBV-ML, respectively. Similar to other citriviruses, CLBV-ML2 has three ORFs. ORF1 is positioned at 67–6024 nt in the genome and encodes a 1986 aa polyprotein, including a viral methyltransferase ; a 2OG-FeII oxygenase ; a peptidase , and an RNA-dependent RNA polymerase 2 domain . ORF2 is positioned at 6022–7110 nt in the genome, and ORF3 is positioned at 7171–8264 nt in the genome, encoding a movement protein and a coat protein, respectively B. Narnaviridae sp. isolate 174-k141_105200 (MZ680060), which was found in pond sediment. Two ORFs were found by the ORFfinder web tool. ORF1 is positioned at 73–789 nt in the genome, and ORF2 is positioned at 725–3991 nt in the genome and possibly encodes RdRp protein. However, no conserved domain was found by the CDD/SPARCLE search  and belongs to the unclassified Virgaviridae (The phylogenetic tree is based on the RNA-dependent RNA polymerase (RdRp) amino acid sequences of representative members of the aviridae A. We proaviridae B.Citrivirus genus show that CLBV-ML2 identified in the present study falls in the clade where sequences of CLBV-PY2 and -PY1  . They coCitrivirus [Prunus spp. [The International Committee on Taxonomy of Viruses recognizes the Citrus leaf blotch virus (CLBV) as the sole species in the genus trivirus . The natnus spp. , Actinidnus spp. , peony [nus spp. , and mulnus spp. . Studiesnus spp. . CLBV venus spp. , and CLBnus spp. . In our nus spp.  but diffIn the ensuing decades, researchers focused on discovering and eliminating viral threats to plant and animal health. However, viruses are not merely agents of destruction but essential components of global ecosystems. Due to the increasing interest in organic farming to reduce the use of synthetic chemicals in agriculture, viral-based vector to manage crop pests and diseases in a sustainable way is a good strategy. A virus-mediated transgenic system is a quick system to deliver genes without any need for transformation and regeneration techniques and has currently been successfully used in apples and other woody species ,40,41,42In this study, we collected eight groups of mulberry leaves from different provinces in China. Three viruses were identified based on genome and phylogenetic analysis, named Citrus leaf blotch virus isolate mulberry alba 2 (CLBV-ML2), Mulberry-associated virga-like virus (MaVLV), and Mulberry-associated narna-like virus (MaNLV).


The number of express boxes worldwide exceeded 170 billion in 2021, and, from several regions in China, tested positive. Therefore, it is important to study the transmission of viruses through express boxes. In this paper, we establish a model of express box virus transmission based on comprehensive consideration of environmental factors, such as temperature, disinfection, humidity, virus release intensity, and volume of vehicle, to study the transmission of express box virus, and explore the spatial and geographic spread variation of express box viruses in China. Several important findings emerged from the study, including: (1) Disinfection can prolong the spread of viruses in the express box for ≥21 h; (2) For every 1 °C rise in temperature, the infected time can be prolonged by ≥1.2 h, and for every 10% rise in relative humidity, the virus transmission time can be prolonged by ≥1.32 h; (3) In an environment suitable for virus transmission, when loaded with 1000, 2000, 4000 express boxes, areas where the express delivery time exceeds 22.56, 18, 14.64 h will face the risk of all the boxes in the carriage being infected. These findings could help public health departments prevent the risk of virus transmission from express boxes. In the past two years, pandemics such as COVID-19 have had serious negative public health and socioeconomic impacts on countries, and the spread of such infectious diseases has slowed the pace of human social progress. To better prevent such pandemics, research literature on COVID-19 is emerging, and scholars from different countries are studying the impact of COVID-19 from their own perspectives, which will help to address social issues during epidemics in various countries. In the past year, there have been many cases of positive tests for the coronavirus in express boxes or infected courier service workers in several Chinese provinces, and China is currently firmly implementing a dynamic zero prevention and control policy to strictly prevent the spread of the coronavirus. Based on the above, and in order to strengthen the research results in the field of public health, this paper explores virus transmission from a new perspective, conducts research in the field of express box virus transmission, and discusses the corresponding recommendations for disease prevention.It is known that the virus is affected by the environment and will show different transmission trends under different conditions. In a study of the effect of temperature on COVID-19 transmission in 427 cities worldwide, Wang M found that the cumulative number of confirmed cases peaked at 8.72 °C, each 1 °C increase in mean temperature in low temperature areas was associated with a 0.83 increase in the number of confirmed cases in cities, and the number of urban confirmed cases decreased by 0.86 cases for each 1 °C increase in average temperature in hot areas ,6,7,8. CThe study of how COVID-19 is transported by express cartridges leading to spatial spread is most critical to understand the mechanism of coronavirus transmission. Coronavirus is transmitted mainly through droplets or aerosols in close contact with an infected person ,15. HoweIn summary, the study of virus transmission in express boxes, and its spatial and geographic spread by delivery, has a practical and scientific significance. Therefore, in this paper, based on the characteristics of express box transportation, the express box infectious disease model is established based on the comprehensive consideration of environmental influencing factors, such as temperature, disinfection, humidity, virus release intensity, and express vehicle transportation volume. Subsequently, the model calculation results are combined to explore the changes of virus spatial and geographical spread due to express box transportation, with the aim of providing prediction support and theoretical suggestions for virus prevention in public health-related fields, to better cope with future infectious disease pandemics.S) latent population (E), infected (I), and cured/recovered (R). Further, susceptible people have the probability β to be infected, susceptible people transform into infected people according to the rate α , and infected people have the probability γ to recover into cured/recovered people. During the pandemic, many scholars have used the above two models to predict the development trend of COVID-19 [Theoretical knowledge related to infectious disease dynamics is the basis of this study, which is a mathematical model of infectious diseases established by Kermack and McKendrick through mathematical and kinetic knowledge . InfectiCOVID-19 ,26,27. TCOVID-19 ,28, alsoCOVID-19 , to studS is the susceptible box, I is the infected box, and the susceptible box has the probability β to be infected, and N is the total number of boxes in courier transportation). Notably, this model has no cured/recovered (R), and combined with the characteristics of virus fomite transmission, there are two reasons: (1) R in the original model represents patients who have recovered, and the virus cannot be detected. However, the coronavirus, when attached to a high-touch surface such as an express box, will have a relatively strong activity and transmission capacity to spread to new individuals within tens of hours [First, in this paper, we establish the express box virus transmission model, Equation (1) , that affects the activity of express box virus transmission was constructed. The environmental impact value ρ, of the express box virus, as a parameter that combines the impact of multiple virus transmission, can help researchers set different environmental conditions in the simulation to achieve the recovery of the real virus transmission environment.Secondly, it was learned through a literature review that the activity of indirect transmission of coronaviruses on the surface of objects diminishes with time, and that this change in the possibility of decline depends on the influence of surrounding environmental factors . After tExpress box dynamic model:Environmental Influence:The factors in Equation (2) have the following characteristics in this study:T = Temperature. In this paper, through the investigation of the express transportation environment, we found that the average temperature in the express carriage is usually kept between is 15~25 °C, there are also special cases, such as in low temperature areas and night driving, the lowest 10 °C, and the highest temperature appears in the summer daytime, the temperature can reach 30 °C. Therefore, the range of values of temperature T in this model is set from 10 °C to 30 °C, and the virus propagation activity decreases as the temperature increases.D = Disinfection. Standard disinfection of carriages as well as express cargo at the time of departure, which will determine the base number of viruses in transport, is divided into two cases in this paper, sterilized and unsterilized.o = Virus release intensity. In this study, free propagation without intervention.V = The volume of the carriage of the express box delivery vehicle.H = Relative humidity. This paper draws on the above-mentioned literature to define the range of humidity in different environments and integrating the range of 40–60% of indoor health humidity and the relative humidity in the closed container truck can reach 95%, set the model minimum relative humidity of 35% (the virus has high activity in dry environment), and gradually increase the relative humidity. The virus activity decreases until the virus activity disappears rapidly when the RH is 95%.ρ of the infected courier box (I) and the susceptible box (S) in the vehicle is proportional to both vehicle travel time and transmission density [b is a constant coefficient proportional to that obtained by fitting the actual data). In combined Equations (1)–(3), can be found the number of individuals in the car infected with express boxes.Finally, according to the knowledge related to infectious disease kinetics, it is known that the infectious rate  density , then coV = 50 m3 of medium-sized vehicles of common express vehicles. The primary COVID-19 free dispersal intensity o = 0.075. In this study, it was assumed that only one delivery box was attached to a sufficient number of highly viable viruses at the time of departure of the delivery vehicle, I (o) = 1. The temperature T, inside the vehicle was set in the range of 10–30 °C. The base relative humidity RH was set at 35% and gradually increased by 20% until it reached 95%. Similar numerical settings were made in the spatial geographic spread study with full consideration of realistic conditions . The viral transmission space was set as the loading volume nditions . The aboT was from 30 °C to 10 °C. It can be seen from the figure: With the increase of temperature T value from 30 °C to 10 °C, the number of infected boxes at the same time are increasing, from the beginning of T = 30 °C to infect 1000 cargoes requires 2.165 days (51.96 h), to T = 10 °C, only requires 1.275 days (30.6 h). Compared to the former, shortened 20.36 h. It shows that the number of infected boxes in the car tends to decrease as the temperature increases.T from 30 °C to 25 °C, the number of infected express boxes does not exceed 10, in a 3-day period. When the T from 25 °C to 10 °C, the number of infected boxes gradually increased, from T = 25 °C, when the infection of 1000 pieces requires 2.93 days (70.32 h), to T = 10 °C, when the infection of 1000 pieces only requires 2.18 days (52.32 h), shortened 18 h.After comparing RH change in the vehicle on virus transmission when loaded with 1000 pieces. T inside the vehicle is the least suitable for virus transmission during the driving process, when T = 30 °C, and changes with the increase of RH of ambient relative humidity . As can be seen from RH increased. In this condition, the model assumes that the entire compartment with 1000 boxes is infected only when the relative humidity RH = 35%, it takes 2.73 days (65.52 h) for the entire boxes to be infected, but with RH ≥ 55%, the number of infected boxes does not exceed 10 in 3 days.RH on the risk of virus transmission in the car, when the car is not disinfected and the temperature inside the car is optimal for virus transmission, T = 10 °C. It can be seen from RH increases from 35% to 95%, all the boxes are infected, from the fastest need of ≈0.94 days (22.56 h), to the slowest need of ≈1.27 days (30.48 h), the time is reduced by 7.92 h. At RH = 35%, it takes 2.73 days (65.52 h) in T = 10 °C and RH = 35%. As the number of boxes increases, the infected time accelerates, and the amount of boxes and the time are inversely proportional. From 49.44 h for infecting 100 boxes to 14.64 h for infecting 4000 boxes, shortened 34.8 h.The corresponding simulation results can be further summarized through Disinfection. Disinfection directly affects the original number and transmission rate of viruses in the express box, its impact rate on the model ≥ 71%.Temperature. As express transportation needs to experience day and night, different temperatures have a large impact rate on the virus, in the model the temperature impact rate ≥ 17.5%, per temperature change ± 5 °C.RH, has an effect rate of ≥23.4% on the model.Relative humidity. Each 20% increase in Boxes. From As shown in First, it is known from Secondly, based on this safe time, combined with the actual express box transportation, we can infer that the virus in the courier car compartment only starts to break out at the specific range covered by the car in a straight line, from which we can assume: the safe time before the virus outbreak as the radius (r) of the best prevention area in the spatial and geographical spread of the express box virus. Further, it is derived that the safety area for express box virus propagation is shown in It is reported that in 2020, China’s courier service companies’ annual business volume was of 83.36 billion pieces, an increase of 31.2% a year, nearly 60 pieces of express boxes per capita, about twice the global average. In 2021, China’s courier companies completed 108.03 billion, an increase of 29.9%. Further, the first confirmed local cases of Omicron were found in Beijing and Shenzhen in the express box cases released by China News in January 2022. Such a large base of courier boxes, combined with the fact that there have been cases of express boxes infected by COVID-19, suggests that the express box sector faces a relatively serious infectious disease prevention challenge.We selected the national courier business volume data of China in 2020  and found that the number of courier boxes in Guangzhou and Zhejiang accounted for more than 48% of the courier sending and receiving data in 31 provinces and that using Guangzhou and Zhejiang as references to study the spread of the express box virus within 31 provinces, can effectively reflect the real situation. Combined with part 2.2 of the article, select 1000, 2000, 4000 boxes as the volume of transported goods, with 22.56, 18, 14.64 h as the time point when all the express boxes are infected during transportation, combined with the actual express transport vehicle driving route (the network navigation software as the transport speed and time), assuming that the express transport vehicle from Guangzhou and Hangzhou departure, set the provincial express unit as the destination, vehicle travel average time of 80 km/h. In which, the transport environment is divided into two categories: (1) environment suitable for virus transmission ; (2) environment unsuitable for virus transmission . Additionally, using the time of arrival of the express box in the province as the time of infection, and marking the color when the province is infected. The spatial and geographic extent of the express box virus infection through transportation is predicted, as shown in In First of all, after the survey and research on the courier field, it was found that the global courier volume showed a gradual upward trend, especially after 2020, when people’s willingness to purchase online increased due to various restrictive precautionary measures for COVID-19 enacted by many countries. Express boxes have the characteristics of large number, frequent contact with humans, and relatively strong cross-regional, which are likely to become an important channel for virus transmission, and there have been reports from countries that have tested positive for COVID-19 in express boxes. Therefore, facing the risk of infectious diseases that may come from courier boxes, this study wants to improve the perception of risk by analyzing the changes in the transmission of viruses from express boxes, and to promote to the health sector to strengthen preparedness and improve response measures .ρ, we wanted to understand the trend of virus transmission between objects that are frequently touched in human daily life such as express boxes, and further predict the impact of virus transmission in express boxes by combining spatial geographic transmission. However, for modeling studies of virus transmission through fomites and thus infection of humans, we recommend reconsidering the candidacy impact factor because of the change in the primary environment (from a closed to an open environment) and it is possible that the probability of virus infection varies from person to person due to different protective measures for each person (wearing masks and hand disinfection), which requires additional settings on an experimental basis.Second, for virus transmission in fomites like express boxes, we need to be clear, while direct transmission is important, the model suggests fomites can also transmit, which is important for exposures that are not in-person. Therefore, fomite transmission may be an important source of risk . Media, The practical significance of this study is that if the COVID-19 virus is found in the express boxes in a certain area, we can track the relevant batches of boxes, combined with the express box virus transmission model, infer the spatial and geographic range of infection by the express box virus, and make targeted prevention to improve the accuracy of virus prevention.Admittedly, our work suffers from three major limitations: First, the comprehensive tests of environmental influences in this study need to be improved; for example, this paper sets out to study the changes of temperature and disinfection on virus transmission in express boxes with other factors held constant. As we know that the environment is a complex system, this study can only choose a constant value research method for the simulation test. Second, there is a deficiency for the combination of the express box virus model and Chinese spatial geography; this study sets the express box departure to the provincial point as the total transportation distance, and the infection of the express box in the carriage when it arrives at the province represents the infection of the whole province, and the study of the change of the number of express boxes in the carriage after the express is redistributed through the provincial transfer station needs to be further explored. Third, this paper takes values based on the initial COVID-19 virus release intensity, while for the continuously mutating virus, the virus release intensity keeps increasing, which will increase the risk of virus transmission and spread, which will require changes in future studies.The main contribution of this study is to explore the spread of COVID-19 in express boxes and derive the trend variation of virus transmission among boxes, and then combine the virus transmission model with Chinese reality to explore the impact and prevention of the express box virus in spatial and geographical spread. Overall, the environmental factors of express box virus transmission are within the controllable range and focusing on the prevention of environmental factors in the carriage during express box transportation can effectively reduce the transmission of the express box virus. For example, courier transport companies can install ventilation equipment in vehicles and maintain a better environmental level in express box storage compartments by monitoring temperature and humidity measuring instruments, which not only helps in the prevention of infectious diseases, but also helps in the protection of products. In addition, it is important to disinfect the environment at the time of shipment, and if long-distance distribution is required, the risk of virus transmission can be reduced by increasing the frequency of disinfection.For individuals, when we sign for a delivery, we touch the delivery box packaging, and then we may face a certain degree of indirect fomite transmission, The best personal prevention method is to dispose of the express box packaging after we sign, do not carry it or store it at home, and after signing, we must disinfect the touching parts—hands.


Peptides with affinity for exosomes were conjugated to the propargyl group of the bottom P(HEMA-co-PgA) segment of the copolymer via a click reaction. The prepared copolymer-grafted beads were characterized by elemental analysis, X-ray photoelectron spectroscopy, scanning electron microscopy, transmission electron microscopy, gel permeation chromatography, and the turbidity of the polymer solution. Results indicated that the copolymer and peptide were successfully modified on the silica beads. Exosomes from SK-BR-3 cells, a human breast cancer cell line, were selectively captured on the prepared beads at 37 °C, as the upper PNIPAAm segment shrank and the affinity between the peptide and exosome was enhanced. Upon lowering the temperature to 4 °C, the captured exosomes were released from the copolymer brush because of the extension of the PNIPAAm segment that reduced the affinity between peptides and exosomes. These findings demonstrated that the prepared copolymer brush-grafted silica beads can capture and release targeted exosomes via temperature modulation. Taken together, the developed copolymer brush-grafted silica beads would be useful for the separation of exosomes using simple procedures such as temperature modulation.The existing methods for exosome isolation, such as ultracentrifugation, size exclusion, and affinity separation, suffer from some limitations. Herein, we aimed to develop temperature-modulated exosome-capturing materials using thermoresponsive polymers and peptides with affinity for exosomes. Poly(2-hydroxyethyl methacrylate-  •Functional beads were developed using thermoresponsive polymer and affinity peptide.•Exosome selectively captured on beads through affinity between the peptide and HER2.•Captured exosome was released from the beads via extension of the polymer.•Temperature-modulated capture of exosome was achieved via temperature change. In advia ultracentrifugation requires large and expensive devices. In contrast, compared to separation methods based on the density and size differences, affinity separation using antibodies exhibits relatively high selectivity. However, for exosome recovery, these methods require the reduction of the affinity between exosomes and antibodies by changing the buffer solution and thus changing properties of exosome membrane proteins. Thus, new exosome purification methods with simple procedures and high selectivity are desired.Exosomes need to be purified to be utilized as diagnostic markers and drug carriers. Various methods, including ultracentrifugation, size exclusion, and affinity separation, have been investigated to isolate exosomes and various isolation kits are commercially available  [[7], 8. HoweverN-isopropylacrylamide) (PNIPAAm) shows thermoresponsive hydrophilic and hydrophobic changes, attributed to hydration and dehydration as well as temperature-modulated extension and shrinking amine (Meco-PgA) brush via a second ATRP. During polymerization, NIPAAm length was modulated by changing the NIPAAm monomer concentration to 500 and 1500 mM. For the modification of long PNIPAAm, NIPAAm  was added to a mixed solvent of 2-propanol (46 mL) and water (4 mL). Dissolved oxygen was removed by bubbling with argon gas for 20 min CuCl2 , ascorbic acid , and Me6TREN  were added to the solution. P(HEMA-co-PgA)-grafted silica beads (1 g) and α-chloro-p-xylene  were then added to the reaction solution under an argon gas atmosphere. ATRP was performed by continuously shaking the bead suspension at 25 °C for 16 h. Next, the bead suspension was filtered, and the beads were washed with acetone and dried at 50 °C for 3 h. The reaction solution was dialyzed as mentioned earlier. The purified polymer solution was lyophilized. The obtained P(HEMA-co-PgA)-b-PNIPAAm-grafted beads were named as “HxPy-Nz,” where z is the millimolar concentration of NIPAAm in the ATRP.The thermoresponsive polymer PNIPAAm was grafted onto the P and N3-Gly-Gly-Gly-Lys-Cys-Cys-Tyr-Ser-Leu (N3-GGGKCCYSL), were used as affinity peptides; these peptides are used as peptide imaging agents with HER2 specificity amine DMSO solution , copper (II) sulfate pentahydrate aqueous solution , and sodium ascorbate water solution . The prepared P(HEMA-co-PgA)-grafted beads (400 mg) or P(HEMA-co-PgA)-b-PNIPAAm-grafted beads (400 mg) were then added to the mixture. The click reaction proceeded with the continuous shaking of the bead suspension at 25 °C for 18 h. The bead suspension was filtered using a membrane filter; beads on the filter were washed with water and acetone and dried in vacuo at 25 °C for 2 h. The peptide-modified beads were named “HxPy-Nz-LTV” and “HxPy-Nz-KCC” and were modified with GGGLTVSPWY and GGGKCCYSL, respectively.Peptides with affinity for exosomes were modified to the propargyl group of the copolymer brush via copper-catalyzed azide alkyne cycloaddition (CuAAc), a click reaction ,79. Two cificity  and shou2.2via elemental analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), gel permeation chromatography (GPC), and the turbidity of the polymer solution.The polymer-grafted beads were characterized To estimate the amounts of initiator, polymer, and peptide, CHN elemental analysis of the prepared beads was conducted using an elemental analyzer . The equations for the estimation are described in the Supplementary Materials.co-PgA)-b-PNIPAAm brush with affinity peptides was observed using XPS . The bead surface was observed using TEM , and bead morphology was observed using SEM .The surface elemental composition of the P, with two serially connected GPC columns  using The phase transition behavior of the polymer was observed from the temperature-dependent turbidity change of the polymer solution. The polymer solution (5 mg/mL) was prepared using phosphate-buffered saline (PBS). The transmittance of the polymer solution at 600 nm was observed with increasing temperature (0.1 °C/min).2.3g and 4 °C for 18 h, and the supernatant was used for the cell culture. We confirmed that the prepared culture medium did not contain exosomes by using dynamic light scattering .The temperature-modulated capture of exosomes was performed using the prepared copolymer-grafted beads with affinity peptides. Exosomes were obtained from the culture medium of SK-BR-3 and HeLa cells. SK-BR-3 cells or HeLa cells were cultured in McCoy's 5A (Modified) medium containing 10% FBS, 1% NEAA, and 1% PS. Ordinarily, FBS also contains exosomes, leading to the contamination of cell-derived and FBS-derived exosomes. To remove exosomes from FBS, FBS was subjected to ultracentrifugation at 100,000×g for 30 min. The culture supernatant was collected, and half the volume of Total Exosome Isolation kit was added. The medium was then gently stirred and incubated at 4 °C for 18 h. Next, the suspension was centrifuged at 10,000×g and 4 °C for 60 min. The supernatant was then removed, and 500 μL of PBS was added. The suspension was subjected to centrifugation filtration using a 0.2 μm-pore diameter filter and the filtrate was used as an exosome suspension. The exosomes obtained in the suspension were confirmed via DLS  to observe particles with 30–100 nm diameter. Ordinarily, exosome characterization is performed by confirming various exosome markers such as CD9, CD63, and CD81  from the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Infectious laryngotracheitis is a very important respiratory disease of poultry. It causes inflammation of the upper respiratory tract and mucous membranes of the eye. A large number of birds in the flock can be infected and mortality is variable. It is a viral disease, transmitted horizontally in a flock and causes great economic losses. In this study, we presented data on the occurrence of the disease after 20 years in unvaccinated flocks in the Vojvodina region and proposed a strategy for the prevention and control of this disease. Infected flocks had a severe clinical symptoms with a decrease in egg production and increased mortality. Clinical symptoms lasted from two to four weeks. We prevented the spread of the virus in one flock applying emergency vaccination with live vaccine. It can be concluded that the disease can easily spread between unvaccinated flocks. The most important thing is to improve biosecurity measures on the farm and implement vaccination in endemic areas.Gallid alpha herpesvirus-1 (GaHV-1), which is transmitted horizontally and most susceptible are chickens older than 4 weeks. After almost two decades since last appearance of this disease in Vojvodina, an outbreak occurred from April 2020 to August 2021 on five laying hen farms and one broiler breeder flock farm. Clinical signs were mild to severe respiratory symptoms, unilateral or bilateral head swelling, serous nasal discharge, conjunctivitis and increased tearing. There was a decrease in feed consumption (2.1–40.0%) and egg production (2.7–42.0%), weight loss and mortality increased (0.8–31.5%). Pathomorphological changes were localized in the upper respiratory tract. Total of 200 carcasses were examined; 40 pooled samples were analyzed by PCR, and 40 by bacteriological analysis. ILT virus was confirmed in tracheal tissue samples. Infected flocks were not vaccinated against this disease. Five flocks had coinfection with Avibacterium paragallinarum. Three-to-four weeks after the first reported case in the flock, clinical symptoms had ceased. Future control and prevention strategies will involve the procurement of flocks vaccinated by recombinant vaccine or the registration of live attenuated vaccines and their use during the rearing period.Infectious laryngotracheitis (ILT) is a respiratory disease of poultry characterized by high morbidity and variable mortality. ILT is caused by  Gallid alphaherpesvirus 1, fam. Alphaherpesvirinae) [Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of domestic poultry caused by the herpes virus (virinae) . It is avirinae) ,4. It cavirinae) ,6. The dvirinae) . Severe virinae) ,8. The IAlphitobius genus on poultry farms can contain live viruses at least 42 days after the disease outbreak on the farm. Airflow between farms is one of the described indirect ways of spreading the ILT virus [The disease is widespread and causes serious economic losses in poultry production ,10. ChicLT virus . An epidLT virus ,12. TherLT virus . RecombiAfter infection, the virus replicates in the respiratory and conjunctival mucosa during a 5–12-day incubation period . ExcretiThe pathological finding shows serous, mucous, bloody or even diphtheria exudate in the mucous membrane of the trachea. Microscopically, lesions are characteristic of alphaherpesvirus infection by the formation of syncytia and intranuclear bodies in the mucosal epithelium , which cThe ILT virus has an envelope and is sensitive to heat, ether, chloroform and other lipolytic solvents. Different strains of the ILT virus have different heat resistance . At loweIn the territory of Serbia, the last reported case of ILT was almost two decades ago on the farms of broiler breeder flocks . There iThe aim of our study is to show the data on a new occurrence of the ILT virus in flocks of poultry with respiratory clinical symptoms on farms in Vojvodina, Serbia. Moreover, measures and strategies for the prevention and control of this disease are proposed.In the period from April to October 2020, respiratory clinical symptoms, unilateral or bilateral swelling of the head, conjunctivitis, increased mortality and decreased egg laying was recorded on 5 farms of laying hens and 1 farm of the broiler breeder flock in Vojvodina . The symData about infected flocks  are summarized in Ten carcasses of dead chickens per flock were sampled from all farms and transported to the Scientific Veterinary Institute “Novi Sad” . A macroscopic examination was performed on all birds. After the pathomorphological diagnosis was completed and the ILT infection was suspected, the organs were sampled for further laboratory tests. For ILT virus testing tracheal tissue from all 10 birds was sampled and a one pooled sample was made per each flock. This means that a total of 20 pooled samples were prepared . DetectiTen birds from each flock were humanely euthanized 28 days after the disappearance of the disease symptoms . One pooled sample of tracheal tissue was collected from humanely euthanized birds for molecular testing in order to determine the presence of ILT virus. A total of 20 pooled tissue samples were collected (one from each flock).Avibacterium paragallinarum . Chocolate agar was incubated at 37 °C 24–48 h in an incubator with 5% CO2. Suspected colonies of A. paragallinarum were sieved from chocolate agar to Columbia agar with the addition of 5% defibrinated sheep blood. Firstly, A. paragallinarum colonies were seeded with loop and then secondly, Staphylococcus aureus ATCC25923 was seeded vertically across the lines of seeded A. paragallinarum as a feeder bacteria. Moreover, blood agar was incubated at 37 °C 24–48 h in an incubator with 5% CO2. Pure culture of A. paragallinarum was identified by gram staining and classical biochemical tests, catalase, oxidase and urease, as well as tests for mobility and carbohydrate fermentation.The conjunctival swabs of the poultry eye were seeded on chocolate agar that provides factor V, necessary for the growth of Escherichia coli (E. coli). Finally, E. coli isolates were confirmed by biochemical tests that include oxidase and catalase assays, glucose and lactose fermentation using triple sugar iron agar (TSI), indole production, methyl-red, Voges-Proskauer test and the inability to use citrate as a carbon source (IMViC test).Twenty-five grams of liver and spleen were seeded in 225 mL buffered peptone water  and incubated for 24 h at 37 °C. After incubation, 10 µL of suspension was transferred to McConkey agar per loopful . After overnight incubation of McConkey agar at 37 °C, colonies were obtained that morphologically correspond g, and the supernatant was used for DNA extraction. Viral DNA was extracted using the commercial kits Indi Spin Pathogen Kit  according to the manufacturer’s instructions. The TaqMan-based real-time PCR was conducted using the commercial kit Quanti Tect Multiplex PCR Kit  with the primers and probes that targeted the conserved area of gC gene part of ILTV genome, as described by Callison et al. (2007) [For the detection of ILTV genome presence, tissue samples of trachea of clinically ill/humanely euthanized (Ethics Committee Opinion no. 05-9821) or found dead animals were chopped with scissors into small pieces in a total amount of 0.2–0.3 g per sample. The prepared samples were placed in 2 mL microtubes and homogenized in 1 mL sterile phosphate-buffered saline for 5 min using a Tissue Lyser LT  operating at 50 Hz. The homogenates were then centrifuged for 10 min at 2000× . (2007) . BrieflyA total of 20 birds belonging to the different 20 affected flocks from the 6 farms under study, were selected for histopathologic studies . FollowiIn our study, the first case of ILT infection occurred in April 2020 on a laying hen farm with birds aged 24 weeks (Farm I). The infection occurred in two flocks at the same time. The flocks have not been vaccinated against this disease. At that time, there were no available data on the presence of the virus on the territory of Serbia. Several days after the onset of symptoms in these two flocks, clinical symptoms such as a decrease in egg-laying capacity, a decrease in food consumption and increased mortality appeared in the other 6 flocks. Five days after the occurrence of ILT on the first farm, a suspicion of infection was reported in laying hens in the production and breeding phase on the second farm (Farm II). The age of the flock on this farm was 4, 36 and 57 weeks. The distance between the first and second farms is about 60 km. Clinical symptoms on Farm II appeared 5 days after the entrance of undisinfected and unwashed trucks from the slaughterhouse, which could have been a potential way to bring this viral agent to the farm. One day after the outbreak occurred on Farm II, clinical symptoms were reported on Farm III in the flock in the production. The distance between the second and third farms is about 12 km. Two days after the outbreak occurred on the third farm, the symptoms also occurred on farms IV and V. Farm IV is at the same location as Farm II, while Farm V is at the same location as Farm III. On all five laying hen farms, the hens were kept in cages and it was observed that most hens with developed clinical symptoms were found in the last third of the facility (last cages). Three weeks after the onset of symptoms on Farm V, an ILT infection occurred on Farm VI, in the broiler breeder flock. This farm is about 33 km away from Farm V. It is important to note that the breeder flock on Farm VI underwent interventional vaccination with a live attenuated ILT vaccine  immediately after the onset of clinical symptoms. The vaccine was administrated via eye drops.During several months of examination and clinical observation of 20 flocks of poultry from 6 farms infected with the ILT virus, a total of 200 carcasses of dead birds and 200 carcasses of humanely euthanized birds were examined—40 pooled samples using molecular diagnostics , and 20 pooled organ samples (liver and spleen) and 20 swabs by bacteriological analysis . The datAll the affected flocks in this study were clinically examined. The clinical examination of the flock revealed mild to severe respiratory symptoms including, the swelling eyelids, infraorbital sinuses and adjacent areas, severe nasal discharge, conjunctivitis, increased tearing, panting and stretching of the head and neck with an open beak . A decreAt the postmortem examination, remarkable findings were localized in the upper respiratory tract. Inflammation of the laryngeal and tracheal mucosa, the presence of mucous exudate and yellow caseous plaques in the trachea and larynx were observed as the dominant findings, while the tracheal lumen was obliterated  a–d. CommMicroscopic examination of the trachea revealed inflammation with lymphocytic infiltration. The tracheal lumen is filled with exudate containing fibrin, inflammatory cells (lymphocytes) and syncytial cells a. In mosThe presence of ILT virus was confirmed by PCR in tracheal tissue samples originating from dead individuals with clinical symptoms and established changes on pathomorphological examination. A positive finding was obtained in all flocks .The cessation of clinical symptoms was observed 2–4 weeks after the first reported case in the flock. A repeated analysis for the presence of the ILT virus using the PCR method was performed 28 days after the last reported clinical case of ILT in the flock. The result was negative in all flocks .E. coli predominating in most individuals and A. paragallinarum in several flocks . The recommended measures consisted of raising the level of biosecurity measures on the farm to a high level and daily informing the epidemiological service of the institute about the health condition of the poultry on the farm.ILT virus infection is characterized by respiratory symptoms, and the first clinical symptoms include dyspnea, tearing of the eyes, swelling of the head and neck with an open beak ,20. ThesWith the further development of the disease, increased mortality appeared (0.8–31.5%) and the number of dead individuals largely depended on the condition of the flock, the age of the flock and the period of the production cycle. Up to 9.2% of deaths occurred in breeding flocks, up to 5% in the flocks at the beginning of the production cycle, and up to 31.5% in older flocks in the last phase of the production cycle . AccordiIn our study, the increase in mortality was accompanied by a decrease in egg laying; it fell by 2.7–42.0%, depending on the age of the flock and the condition. Most authors describe the clinical picture with respiratory symptoms, high morbidity and variable mortality, with a significant decrease in egg laying ,15,24. IA. paragallinarum, which indicates a synergistic effect between the ILT virus and this bacterium [A. paragallinarum had a mortality between 0.8 and 31.5%, decreased egg laying by 2.7–31.6% and decreased feed consumption by 2.1–5.5%. According to the data of Jonare et al. (2020), coinfection with A. paragallinarum was the most frequent in flocks infected with ILT [A. paragallinarum can result in a shorter period of incubation and increased mortality [E. coli and A. paragallinarum are common [A. paragallinarum, while the highest decrease in egg laying (42.0%) and decrease in feed consumption (40.0%) were in the flocks that did not have coinfection with A. paragallinarum (Macroscopically, changes in birds infected with the ILT virus are most pronounced in the upper respiratory tract, tracheal and laryngeal mucosa. ILT is characterized by the presence of mucosal exudate and yellow caseous plaques in the trachea and larynx, inflammation of the mucosa, hemorrhagic tracheitis with blood clots and hemorrhagic mucus along the entire trachea ,28,29. Vacterium . In our with ILT . Couto eortality . Moreovee common , which illinarum . CoinfecMicroscopically, the occurrence of a syncytium with eosinophilic intranuclear inclusion bodies is considered a pathognomonic finding in ILT virus infection. Herpesvirus inclusion bodies are clusters of viral particles, proteins and genomes and most commonly occur in severe and moderate forms of the disease . Our stuA. paragallinarum. In our study, it is suspected that the virus entered two farms through the unwashed and disinfected slaughterhouses and rendering vehicles. This shows that the application of strict biosecurity measures on farms can prevent the occurrence of ILT. Future strategies for the control and prevention of infectious laryngotracheitis will include the procurement of flocks vaccinated with recombinant vaccines or the registration of live attenuated vaccines and their use during the rearing period.Infectious laryngotracheitis of poultry is a very important disease for modern poultry, and it is on the list of diseases that must be reported. After almost two decades, it appeared in Vojvodina on a broiler breeder flock farm and laying hen farms. Based on the characteristic clinical picture and pathoanatomical findings, ILT was suspected and the presence of the ILT virus was confirmed using the molecular PCR technique. Morbidity was up to 45%, and mortality was up to 31.5%. The decline in egg production was more than 40%, but in 2–4 weeks it returned to the level of technological norms. Bacteriological analysis in several flocks confirmed the presence of


Tobamoviruses are agriculturally relevant viruses that cause crop losses and have infected plants in many regions of the world. These viruses are frequently found in municipal wastewater, likely coming from human diet and industrial waste across wastewater catchment areas. As part of a large wastewater-based epidemiology study across Southern California, we analyzed RNA sequence data from 275 influent wastewater samples obtained from eight wastewater treatment plants with a catchment area of approximately 16 million people from July 2020 to August 2021. We assembled 1,083 high-quality genomes, enumerated viral sequencing reads, and detected thousands of single nucleotide variants from eight common tobamoviruses: bell pepper mottle virus, cucumber green mottle mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, tomato brown rugose fruit virus, tomato mosaic virus, tomato mottle mosaic virus, and tropical soda apple mosaic virus. We show that single nucleotide variants had amino acid-altering consequences along with synonymous mutations, which represents potential evolution with functional consequences in genomes of these viruses. Our study shows the importance of wastewater sequencing to monitor the genomic diversity of these plant-infecting viruses, and we suggest that our data could be used to continue tracking the genomic variability of such pathogens.IMPORTANCE Diseases caused by viruses in the genus Tobamovirus cause crop losses around the world. As with other viruses, mutation occurring in the virus’s genomes can have functional consequences and may alter viral infectivity. Many of these plant-infecting viruses have been found in wastewater, likely coming from human consumption of infected plants and produce. By sequencing RNA extracted from influent wastewater obtained from eight wastewater treatment plants in Southern California, we assembled high-quality viral genomes and detected thousands of single nucleotide variants from eight tobamoviruses. Our study shows that Tobamovirus genomes vary at many positions, which may have important consequences when designing assays for the detection of these viruses by agricultural or environmental scientists. Tobamovirus, which represents important plant pathogens causing substantial crop losses to the global agricultural industry  of eight tobamoviruses sourced from 275 samples across eight wastewater treatment plants from July 2020 to August 2021 , 15. TheResults. We aligned 156,825,269 quality-filtered, deduplicated, matching paired-end reads  across 275 samples from eight water treatment plants . Of the paired-end reads that mapped to the eight tobamoviruses, 0.34% were bell pepper mottle virus (BPeMV), 12.90% were cucumber green mottle mosaic virus (CGMMV), 11.90% were pepper mild mottle virus (PMMoV), 1.31% were tobacco mild green mosaic virus (TMGMV), 64.06% were tomato brown rugose fruit virus (ToBRFV), 5.6% were tomato mosaic virus (ToMV), 1.87% were tomato mottle mosaic virus (ToMMV), and 2.02% were tropical soda apple mosaic virus (TSAMV) (doi.org/10.7280/D1S69X)  along with the SNVs’ mutational consequence (synonymous or nonsynonymous) in /D1S69X) .Discussion. Wastewater-based epidemiology (WBE) has been used to characterize pathogen abundances and genomics for a variety of diseases and is often employed to detect antibiotic resistance or diseases relevant to public health , CGMMV (NC_001801.1), PMMoV (NC_003630.1), TMGMV (NC_001556.1), ToBRFV (NC_028478.1), ToMV (NC_002692.1), ToMMV (NC_022230.1), and TSAMV (NC_030229.1) and calculated the relative abundance by dividing the number of reads that mapped to each independent virus with reads that mapped to all eight viruses with SAMtools  under BioProject accession number PRJNA729801 and on the Dryad Digital Repository (doi.org/10.7280/D1S69X) (/D1S69X) . We repo


Despite the high HIV associated burden, Mozambique lacks data on HIV counselling and testing (HCT) costs. To help guide national HIV/AIDS programs, we estimated the cost per test for voluntary counselling and testing (VCT) from the patient’s perspective and the costs per person tested and per HIV-positive individual linked to care to the healthcare provider for VCT, provider-initiated counselling and testing (PICT) and home-based testing (HBT). We also assessed the cost-effectiveness of these strategies for linking patients to care.Data from a cohort study conducted in the Manhiça District were used to derive costs and linkage-to-care outcomes of the three HCT strategies. A decision tree was used to model HCT costs according to the likelihood of HCT linking individuals to care and to obtain the incremental cost-effectiveness ratios (ICERs) of PICT and HBT with VCT as the comparator. Sensitivity analyses were performed to assess robustness of base-case findings.Based on costs and valuations in 2015, average and median VCT costs to the patient per individual tested were US$1.34 and US$1.08, respectively. Costs per individual tested were greatest for HBT (US$11.07), followed by VCT (US$7.79), and PICT (US$7.14). The costs per HIV-positive individual linked to care followed a similar trend. PICT was not cost-effective in comparison with VCT at a willingness-to-accept threshold of US$4.53, but only marginally given a corresponding base-case ICER of US$4.15, while HBT was dominated, with higher costs and lower impact than VCT. Base-case results for the comparison between PICT and VCT presented great uncertainty, whereas findings for HBT were robust.PICT and VCT are likely equally cost-effective in Manhiça. We recommend that VCT be offered as the predominant HCT strategy in Mozambique, but expansion of PICT could be considered in limited-resource areas. HBT without facilitated linkage or reduced costs is unlikely to be cost-effective. Timely HIV diagnosis and linkage to care are essential for improved HIV outcomes  but are In Mozambique, where the adult HIV prevalence is among the highest in the world, only 61% PLHIV know their HIV status, and 54% are receiving ART . At the Economic evidence in relation to costs and cost-effectiveness of these HCT strategies in linking PLHIV to care is lacking. By drawing on prospective data from the Manhiça cohort study , we estiIn the literature, unit costs of HCT strategies from the provider’s perspective are derived from program costs and usually reported as average cost per person tested and/or average cost per HIV-positive individual identified. The former is more relevant for our study and shall be examined in detail for VCT, PICT, and HBT. Estimates of unit costs per person tested vary. For example, VCT costs in Kenya, Swaziland, Tanzania, and Uganda were reported to be US$8.27–US$28.93 –17, and In 2012, HIV prevalence and incidence in Manhiça district were estimated to be 40% and 3.6 infections/100 person-years, respectively , 22. TheWe employed a micro-costing (bottom-up) approach  to estimTo determine financial costs, we processed data on capital  and recurrent  costs collected May 2014–June 2015. We utilized time and motion data to determine the financial costs of personnel and transport. Time data tracked the time taken by counsellors to travel to and between participants’ houses during HBT and to conduct each counselling session either at the District Hospital or at each participant’s house. On the other hand, motion data included travelling speeds to determine fuel costs. We did not include training costs because HCT in the cohort study was provided by healthcare workers who were not specially trained.The cost per VCT attendance from the patient’s perspective was determined by a similar micro-costing approach using self-reported explicit costs and implicit costs. Implicit costs were calculated by valuing the patients’ time spent travelling to and waiting at the VCT facility using the average monthly minimum wage in Mozambique, expressed as an hourly wage and assuming an average of 176 work hours (22 days × 8 h/day) per month. We assumed patients would not incur any additional costs for HBT and PICT.A questionnaire was used to elicit demographic information Appendix . From thFor cost estimate analysis, we included data on all individuals who tested HIV positive (n = 1277), including non-enrolled individuals (n = 155) who had received a HIV-positive test result previously in a concomitant study. These 155 patients were tested using the same procedure as with the other participants in the main cohort study and were included in our sample size. Of the 1277 patients, 350 were enrolled from VCT and were included in cost estimates from the patients’ perspective. Time data for the following variables were highly negatively skewed due to measurement errors during data collection: travelling to individual houses, performing the test, waiting for test results, and explaining the results. We deemed the 85th percentile to be an appropriate upper limit to capture a reasonable proportion of these data without including outlying values, replacing observations above this percentile with the value at the 85th percentile. All data analyses were conducted using Microsoft Excel 2016 (V.16.14.1) and Stata (V.13.1: Stata Corporation).We performed descriptive statistical analysis to compute mean (with standard deviation) and median (with maximum and minimum ranges) costs per person tested positive from both the patient’s and provider’s perspective. All costs were expressed in 2015 US$ at an exchange rate of 33.00 Mozambique New Metical (MT) per US$ .The cost of testing each individual was determined by summing costs of each individual resource, obtained by multiplying the quantities of each resource expended with their approximated unit costs Appendix .Implicit costs to patients were calculated by valuing their time spent travelling to and waiting at the VCT facility, using the average monthly minimum wage in Mozambique . CapitalA decision tree model Appendix  was desiThe model captured all steps of the HIV care cascade for both VCT and PICT from diagnosis to retention in care after 12 months of follow-up. For HBT, the model additionally captured steps before the HCT process, from being reached through HBT to obtaining consent. One-year cumulative proportions, reported as the percentage uptake at each step of the HIV care cascade  at each step was modelled; and.Costs incurred for individuals who cannot be reached through or deemed ineligible for HBT are inconsequential to total program costs.The key assumptions of the model were:We calculated the expected average costs and linkage-to-care proportion per person tested for each HCT strategy, by summing costs and outcomes of each branch of the tree weighted by their associated probabilities of occurrence. A ratio of average costs and average expected linkage-to-care proportion was calculated to determine expected HCT linkage-to-care costs.Unlike the derivation of CERs, data only for enrolled individuals was used to determine ICERs to facilitate accurate comparisons of effectiveness. We determined ICERs by dividing the difference in expected costs over the difference in the expected proportion of enrolled individuals linked to care.Our univariate sensitivity analysis varied key cost-related parameters within predefined sensitivity ranges Appendix  to exploWe performed deterministic and probabilistic sensitivity analyses (DSA and PSA) as part of the CEA. In the DSA, probabilities and average costs per test were varied at the maximum and minimum values defined by their respective distributions Table . In the Base-case median costs were higher for VCT per individual tested  than for PICT per individual tested  but were lower than HBT per individual tested . A breakdown of total costs into explicit  and implicit costs  showed most costs (~ 80%) were implicit, which reflected waiting times and travelling times to the hospital  for every individual successfully linked to care. As there was no clearly defined WTA threshold, a CEAC was constructed Fig. . Since tTo our knowledge, this is the first study in Mozambique to estimate costs and cost-effectiveness of VCT, PICT, and HBT. We found the median cost per VCT attendance to be as low as US$1.00, largely comprising implicit costs. This is commensurate with VCT prices and patients’ willingness-to-pay reported elsewhere , 35, sugWe found the average cost to the provider per individual tested and derived costs per HIV-positive patient linked to care to be greatest for HBT, followed by VCT and PICT. Both the magnitude and trend of costs elicited were mostly consistent with the available literature for neighboring SSA countries (in 2009 prices), with the exception of the study by Menzies et al. (in 2007 prices) –15. NotwWe found HBT to be substantially more expensive than VCT and PICT. This was not consistent with the available literature. Five studies across Uganda, Kenya, and South Africa reported HBT costs ranging from US$5.00–US$29.00 per client tested that were in several cases lower than reported VCT and PICT costs , 18–20. CEA results suggested PICT was not cost-effective to link PLHIV to care, relative to VCT; more precisely, PICT was less expensive but also less effective than VCT. However, this result was only marginal. Moreover, the lack of robustness in the base-case results of the CEA comparing PICT and VCT, coupled with the fact that PICT was consistently reported to be cheaper than VCT , 15 yet As expected, HBT was more expensive and had a lower expected linkage-to-care proportion than VCT. This was consistent with several studies , 41–46. Our study had limitations. Because data were only available for PLHIV, we modelled total program costs. The inability to determine real program costs may limit the usefulness of our findings for policymakers . MoreoveResults from this study are hardly generalizable to other countries. Manhiça is a small, semi-rural district in Mozambique where HCT services are offered free-of-charge at district hospitals. It is unclear how the imposition of charges, like in Kenya and Tanzania , 35, wouBecause our dataset lacked detailed cost information, we were unable to evaluate the combination of VCT with either PICT or HBT against standalone VCT. Future studies, however, could investigate such combinations and provide information to policymakers on how best to improve existing HIV-testing policies in Mozambique.A CEA may be unsuitable for assessing the utility of HBT to meet targets for HIV eradication. CEAs are often designed to maximize efficiency at the expense of achieving distributive equality and equity . TherefoDespite these limitations, this study generates, for the first time, information for HIV healthcare policy decision making in Southern Mozambique, a setting characterized by one of the highest community based HIV prevalences in the world . From the research perspective, this project calls for the need to test the cost-effectiveness of comprehensive home-based strategies including not only screening but also linkage to care and promotion of ART adherence. A similar combination prevention approach involving universal HIV testing and treatment has recently been assessed to be a cost-effective strategy at thresholds greater than US$800 per DALY averted in Zambia and South Africa . This ma


Japanese soil-borne wheat mosaic virus (Furovirus) is a damaging pathogen of wheat and barley. This virus can survive in the soil for several decades, so the deployment of resistant cultivars represents the only practical control measure. Here, a genetic analysis has identified two regions of the barley genome—one on chromosome 2H and the other on chromosome 3H—as harboring gene(s) encoding resistance to this virus. The joint presence of both loci, termed Jmv1 and Jmv2, made the plants essentially immune, with resistance being dominant over susceptibility at each locus. Phylogenetic analysis showed that the virus is not closely related to the type Furovirus species Soil-borne wheat mosaic virus. There was a difference between the RNA1- and RNA2-based phylogenies of the virus species in Furovirus implying the independent segregation of the virus subgenomes. Japanese soil-borne wheat mosaic virus (JSBWMV) belongs to the genus Furovirus, a set of rigid, rod-shaped bipartite RNA viruses harboring the two positive sense strands RNA1 and RNA2 (Furovirus genus include Chinese wheat mosaic virus (CWMV), Soil-borne wheat mosaic virus (SBWMV), Soil-borne cereal mosaic virus (SBCMV), Oat golden stripe virus (OGSV) and Sorghum chlorotic spot virus (SrCSV)  . The typ (SrCSV) . The pat decades , while sTriticum aestivum) have identified that the presence of the gene Sbm1 protects the host against SBWMV infection , it has been shown that resistance against SBCMV is conferred by a quantitative trait locus (QTL) QSbm.ubo-2BS which maps to a chromosomal site similar to that occupied by Sbm2 .The mapping population was developed from a cross between cultivar (cv.) “Tochinoibuki” and cv. “Sukai Golden”, both of which are two-rowed malting barleys bred at the Tochigi Prefectural Agricultural Experimental Station  and 6 April 2016 (Rep. #2). Both parents and the set of F7 RILs were sown on 21 October 2016, with leaf sampling timed for 16 March 2017 (Rep. #3) and 4 April 2017 (Rep. #4).Plants were exposed to JSBWMV infection by raising them in a disease nursery at Yawara . Both parental cultivars, their F2HPO4.12H2O, 0.2 g/L KH2PO4, 0.2 g/L KCl, 0.5 mL/L Tween 20). Following centrifugation  in an AR015-24 centrifuge , a 0.1 mL aliquot of supernatant was subjected to a double antibody sandwich-ELISA  was genotyped using a Fluidigm 96.96 Dynamic Array IFC chip , following the SNPtype 96x96 v1 protocol . Polymorphic sites were chosen to identify a population of evenly distributed genetic markers. The resulting linkage map was deduced using JoinMap v4.1 software  was used for QTL analysis. Composite interval mapping was performed using the model 6 forward and backward method of the Zmapqtl procedure implemented in Windows QTL Cartographer v2.5  and its quality checked using a Bioanalyzer device . Only RNA samples displaying a RIN value >7 were used for library construction. The libraries were assembled using a TruSeq Stranded Total RNA Library Prep Kit with RiboZero (Illumina) and sequenced using the NovaSeq 6000 NGS system (Illumina). To remove adaptor sequence, the fastq data generated were processed by imposing the Trimmomatic-0.33 program . The resMultiple alignments of both the RNA nucleotide and the predicted amino acid sequences were performed using the ClustalW algorithm  responsible for the resistance of cv. “Sukai Golden” were dominant and were nuclear rather than cytoplasmic. Of the 284 cv. “Tochinoibuki” × cv. “Sukai Golden” F2 progeny assayed in two independent experiments, 18 proved to be infected and 266 uninfected, consistent with the 1:15 segregation ratio resulting from the presence in cv. “Sukai Golden” of two independent, dominant genes .A representative outcome of the ELISA-based quantification of the JSBWMV titer in the leaf is shown in A set of 7,508,510 paired reads was recovered from cv. “Tochinoibuki” shoot and root tissue and 4,169,883 from cv. “Sukai Golden” shoot and root tissue . After t6 RILs  within a 5.8 cM/6.2 Mbp interval flanked by FB0211  and FB0212 (11.2 Mb) (Jmv2) and FB0245 (591.6 Mb) didn’t recombine (Jmv1 and Jmv2).Linkage mapping using the appropriate genetic markers and ELISA scores in segregating RILs  placed t11.2 Mb) . The phyecombine , so no cecombine , as expeJmv1 and Jmv2 alone provided a partial level of field susceptibility, while RILs carrying both genes in the homozygous state universally escaped infection in 2016 , while RILs carrying both genes were essentially immune. The result implied that the two dominant genes complementarily encode perfect resistance to JSBWMV.Both  in 2016 . In 2017Of the paired reads generated by the total RNA-Seq, 1.0% matched sequences represented in the RNA1 reference sequence, as did 1.7% to the RNA2 reference sequence. There was no between duplicate sequence variation between reads matching the RNA1 sequence, but there was one instance of a single nucleotide polymorphism (T6304A) uncovered between the reference RNA1 sequence LC481573 and sequence LC642564: this variant is predicted to generate a L2102M alteration in the virus’ 37 kDa movement protein. Sequence LC642565 featured heterogeneity at two sites (T346C and A1534G) with respect to the type RNA2 sequence LC481574, with, respectively, the T and A nucleotide matching the reference sequence. However, neither variant is predicted to generate any change at the polypeptide level. The first open reading frame of RNA1 encodes a 152 kDa protein including a putative methyltransferase domain and a helicase domain; its read-through at the leaky opal stop codon results in the translation of an additional 59 kDa RNA polymerase. The second open reading frame encodes a 37 kDa movement protein. The first open reading frame of RNA2 encodes a 19 kDa capsid protein and a 64 kDa read-through protein, while its second open reading frame encodes a 19 kDa cysteine-rich protein. These analyses replicated the results of Furovirus species  is shown in A phylogenetic analysis based on six known Barley yellow mosaic virus act as recessive alleles  of these resistance genes to protect barley against infection by other strains of JSBWMV and even perhaps by other Furovirus species remains to be established.The phylogenetic analysis based on the two viral RNA sequences was able to identify a number of differences between European (SBCMV), Japanese (JSBWMV), Chinese (CWMV) and US (SBWMV) species. The joint presence of both contexts . The joiK.O., T.T., T.Ko, K.N. and T.Ka conceived the experiments, T.Ka developed the germplasm, K.O., K.N. and T.Ka carried out the ELISA experiments, K.S. performed the RNA-Seq analysis, T.T. was responsible for the detection of nucleotide variation and S.F. for genotyping, K.O. and T.O. carried out the QTL mapping, Y.O., K.M. and T.O. performed RNA-Seq on the virus, and K.O., T.T., S.F., Y.O., T.O. and T.Ko wrote the paper.Supplemental FiguresSupplemental Table 1Supplemental Table 2Supplemental Table 3Supplemental Table 4Supplemental Table 5Supplemental Table 6


Dear Editor,Poxviruses are the largest DNA viruses and have numerous genus and species. Generally, they can cause typical skin damage to their infected hosts, including humans and many kinds of animals. In human history, through the mass use of the Vaccinia vaccine, smallpox caused by smallpox virus is the only virus that has been wiped out. Theoretically, the poxvirus vaccine can create solid immunity against viral infection. However, two important poxviruses that originated in Africa have been spreading rapidly on other continents in recent years ,2.The first one is the lumpy skin disease virus (LSDV) belonging to the Capripoxvirus genus, which mainly affects cattle and occasionally infects non-cattle animals, including eland and giraffes . In addiOrthopoxvirus genus. Genetically, MPXV has two clades: the central African clade and the West African clade. The former could cause higher fatality rates than the latter. However, cross-immune protection existed among them. Due to the previous vaccination of vaccinia, MPXV was endemic in several African countries, including Nigeria, Benin, and Liberia.The second one is the zoonotic monkeypox virus (MPXV), which mainly affects monkeys, Gambian pouched rats, and squirrels, and occasionally infects humans, causing small outbreaks. In addition to fever, headache, muscle aches, and lymphadenopathy, distinctive skin rash is typical. Comparing the above LSDV, MPXV, a relatively young virus, was first found in the Democratic Republic of the Congo (DRC), central Africa (bordering Zambia), in 1970. MPXV, the same as vaccinia virus and smallpox virus, belonged to the https://worldhealthorg.shinyapps.io/mpx_global/). The evidence indicates that MPXV is quickly going global [With the eradication of the smallpox virus, the cessation of smallpox vaccination in the 1980s lead to a specific immunity decline in humans. In 2017, the big West African MPXV outbreak occurred in Nigeria. Since then, MPXV jumped to Singapore (Southeast Asia), Israel (West Asia), and the United Kingdom (Europe). Incredibly, from 1 January to 1 September 2022, 51,163 confirmed cases of monkeypox and 17 deaths have been reported to WHO from 102 countries/territories/areas in six WHO Regions A and B  from the Department of Agriculture and Rural Affairs of Guangdong Province, and the grant (No. 201127186621161 to S-LZ) Haojiang District Science and Technology Bureau.This study was supported by the following grants: the grant (No. 2021B1212050021 to Z-HX) from the Shao-Lun Zhai, Ming-Fei Sun, Zhi-Hong Xu are the co-first authors. Shao-Lun Zhai, Ming-Fei Sun, Zhi-Hong Xu collected the data and drafted the paper for the work. Gang Wang, Chun-Ling Li, Chunfu Zheng and Ming Liao review the paper. Shao-Lun Zhai and Zhi-Hong Xu did the financial support, review, and final approval of the paper to be published. All authors read and approved the final manuscript.The data used and analyzed during the current study are available from the corresponding author on reasonable request.The authors have no competing interests to declare.


The burden of bronchiolitis is remarkable due to high morbidity in infants. The aim of this study was to evaluate bronchiolitis‐associated costs for patients' families and the healthcare system.This retrospective, descriptive study included 136 infants under 12 months of age treated at Tampere University Hospital, Finland, between October 1, 2018 and March 31, 2020, with bronchiolitis as the main diagnosis. The data consists of patient background and medical information and of estimated costs for the families and for the healthcare system. The data were collected from the hospital's electronic patient files and registries and were analyzed with descriptive statistical analyzes using SPSS v. 26 software.The total median costs associated with bronchiolitis from the perspective of families and healthcare were €16,205 per patient if intensive care was needed and €2266 per patient treated only on the ward. The median costs for the families were €461 and €244, respectively, and for the healthcare system, they were €15,644 and €2019.The majority of the total costs for treatment were due to healthcare costs and only 10% of costs were targeted at families. Bronchiolitis‐associated total median costs were 7.2 times higher and the families' costs were 1.9 times higher if intensive care was needed instead of treatment on the ward only. According to one German study, total socioeconomic costs were €3700 per hospitalization and the parental cost was €87.The aim of this retrospective study was to evaluate costs associated with bronchiolitis hospitalization from the perspectives of families and healthcare organizations, that is, the hospital, as well as to separately evaluate the costs for treating infants in PICUs or on wards only.2This was a retrospective chart review describing the cost of bronchiolitis hospitalization in infants treated at Tampere University Hospital (Tays), Finland. Tays provides secondary and tertiary healthcare for ~4400 infants under 1 year of age living in Tampere and its surroundings, both in urban and rural areas. The healthcare system in Finland is universal and tax‐funded. The copayment for the patients or the families is low, meaning that healthcare services are affordable for all patients. Public or private primary healthcare provides only preventive or outpatient healthcare for children, which means that all patients in this area who need inpatient care are treated in Tays.All infants under 12 months requiring inpatient treatment between October 1, 2018 and March 31, 2020, for bronchiolitis  were included in the study. Bronchiolitis was defined as the first expiratory difficulty during an acute viral lower respiratory tract infection in infants under 12 months of age. The data were collected from electronic patient files and registries at Tays.One of the authors (EM) collected the background and medical data in a structured case report form, including the details of gender, age on admission, number of siblings, length of stay (LOS) in days on the ward and in the PICU, number of family visits during hospitalization, the distance of travel between hospital and patients' home, private doctor visits, ambulance transfers, primary and secondary emergency department (ED) visits and readmissions within 4 weeks of the index hospitalization.The cost data consists of the costs from the perspectives of families and healthcare, that is, the hospital. The estimate of families' costs included hospital fees , travel costs, additional meal costs due to hospitalization, private doctor fees, and ambulance fees; children's visits to primary healthcare facilities are free of charge in Finland and thus were not included. The detailed cost estimations for the families are presented in Table The healthcare costs were defined as the costs and reimbursement caused by the hospitalization of bronchiolitis and were collected from Tampere University Hospital's cost‐per‐patient software. The detailed costs consist of daily costs of treatment, laboratory charges, imaging charges and other costs due to treatment. In Finland, hospital reimbursement is paid by the municipalities.The costs are presented in Euros (€). Costs were not adjusted for inflation due to the short study period (19 months) and low inflation during that time. When comparing results to earlier studies, the annual mean exchange rate for the publication year was used: €1 = $0.8956 (in 2001), €1 = $0.9456 (in 2002), and €1 = $1.3281 (in 2013).This study was performed with the permission of the Research Director of the hospital. As the study is based on hospital registers and patients were not contacted, no approval from the ethics committee was required.2.1SPSS statistics version 26 (IBM Corp) was used to perform the descriptive analysis. The data were presented as numbers and percentiles. In addition, nonnormally distributed continuous data were presented with median and either interquartile ranges (IQR) or minimum and maximum values. Spearman's correlation was used to analyse the correlation between hospitalization costs and the LOS.3A total of 136 infants under 12 months of age treated between October 1, 2018 and March 31, 2020 for bronchiolitis were included in the study. All patients presented at the ED, except one who was admitted directly to intensive care from a secondary care hospital . Most of the patients were boys (53.7%) and the most common number of siblings was one . The median reimbursement was lower than the median costs both for those treated in the PICU and those treated only in the ward. Most of the hospitalization costs consisted of the daily cost of treatment, with other relevant cost sources being imaging and laboratory costs. Median imaging costs were €56 , with imaging being conducted for 31 infants. Laboratory costs were higher among infants treated in the PICU than among those treated on the ward: €1155 versus €70, respectively  and the rest were due to families' costs  and due to employers .Our results support previous evidence that family costs associated with bronchiolitis hospitalization are a considerable expense for families. The median family cost was €244 per hospitalized infant with bronchiolitis treated on the ward and €461 per hospitalized infant with bronchiolitis treated in the PICU. However, the family costs ranged up to €1000 depending on the LOS in hospital and the distance of travel. Total travel expenses formed a substantial part of total family costs, alongside hospital fees. Direct comparison of family costs with those of previous studies was not possible because of the different parameters used.In a French retrospective cohort analysis including 525 infants requiring respiratory support, researchers found that children treated in the PICU between 2006 and 2010 because of bronchiolitis with nasal continuous positive airway pressure had mean hospital costs of €16,800.Previous findings are in line with our results that the median hospitalization costs were €2019, €15,644 and €2422 for patients treated only on the ward, for patients treated in the PICU and for all patients, respectively. The hospitalization costs seem to have increased in our hospital since 2015, which may be partly due to inflation or changes in both internal and external prices, but also perhaps due to a different approach used in the evaluation of costs in the studies. As shown in the present study, the reimbursement is lower than hospitalization costs, which is mostly the result of political decisions. Differences between earlier studies may be explained by differences among healthcare organizations and healthcare financing in countries as well as by different ways of estimating the costs in the studies. However, the results are remarkably different when comparing the costs between the high‐income and low‐income countries,Previous studies have found that by following the local or well‐established bronchiolitis guidelines, both the costs associated with bronchiolitis hospitalization and the use of unnecessary—and often harmful—treatments have decreased. A Finnish study published in 2018 demonstrated a substantial decrease in costs related to bronchiolitis medications after the publication of national Current Care Guidelines advising against racemic adrenaline use in treatment.5The main limitation of our study was its retrospective nature. Families' costs were probably underestimated due to the study design; not all the information needed was available from electronic patient files. As an example, the information regarding parents' working status was unavailable, and consequently, loss of income could not be estimated. Moreover, we could not estimate the need for supplemental childcare, which could sum up to a significant cost for parents. In addition, we did not have information on parents' money consumption and, accordingly, the meal costs were most likely underestimated. For lower‐income families, in particular, having a child hospitalized can be a substantial extra expense. On the other hand, we tried to estimate as well as possible every cost associated with bronchiolitis treatment, for example, travel allowances and meal allowances. We did not use any validated questionnaires to extract data from the hospital registers, but all data that was available was collected. Thus, this study adds necessary information about costs from the family's perspective.ad hoc analysis by removing this patient, and the results did not change.Another limitation is that the number of patients treated in the PICU was relatively small. Moreover, one of the patients had significantly higher costs than the others due to a rare underlying condition and very expensive treatment needed during an extended hospitalization. To minimize the effect, we used the median value instead of the mean value. In addition, we also verified this in an 6The majority of the bronchiolitis‐associated costs were due to healthcare costs both in those treated in the intensive care unit (97%) and for those treated only on the ward (89%). Bronchiolitis‐associated total median costs were 7.2 times higher if intensive care was needed instead of treatment on the ward only. Approximately 10% of the total costs of all patients were targeted at the families, and the families' costs were 1.9 times higher if intensive care was needed.Design and conceptualization: Paula Heikkilä and Sauli Palmu. Formal analysis: Emilia Mäntynen and Paula Heikkilä. Interpretation of the data: Emilia Mäntynen, Sauli Palmu and Paula Heikkilä. Funding acquisition: Paula Heikkilä. Writing–review and editing: Sauli Palmu and Paula Heikkilä. Writing–original draft: Emilia Mäntynen. All authors have read and approved the final version of the manuscript.The authors declare no conflicts of interest.This study was approved by the Research Director of the Tampere University Hospital (R19631S). According to Finnish law, neither approval from the Ethics Committee nor informed consent from the subjects and/or their guardians were not required for this retrospective register‐based study. This study was carried out in accordance with the Declaration of Helsinki. All patient data were managed and conformed to the European Union's General Data Protection Regulation (GDPR) and the data security legislation of Finland.Paula Heikkilä affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained.Supporting informationClick here for additional data file.