Capsicum annuum L.) plants produce berry fruits that are used as spices. Here, we examined the viromes of 15 pepper cultivars through RNA sequencing. We obtained 1,325 virus-associated contigs derived from 8 virus species. Bean broad wilt virus 2 (BBWV2) and cucumber mosaic virus (CMV) were identified as the major viruses infecting pepper plants, followed by potato virus Y, bell pepper endornavirus, and hot pepper endornavirus. The proportion of viral reads in each transcriptome ranged from 0.04% to 24.5%. BBWV2 was the dominant virus in seven cultivars, whereas CMV was dominant in five cultivars. All the bell pepper cultivars showed severe viral disease symptoms, whereas the commercially developed hot pepper cultivars were asymptomatic or had mild symptoms. In addition, 111 complete viral segments were obtained from 7 viruses. Based on the obtained viral genomes, the genetic relationship between the identified viruses and quasispecies of BBWV2 and CMV in each pepper plant was determined. Newly designed primers for nine viruses confirmed the results of RNA sequencing. Taken together, this study, for the first time, provides a comprehensive overview of viromes in 15 major pepper cultivars through RNA sequencing.Pepper ( Capsicum annuum L., a member of the nightshade family Solanaceae, is known to produce berry fruits [The pepper plant y fruits . Peppersy fruits . The play fruits . Many peIn Korea, bell peppers and hot peppers are widely cultivated. Hot peppers are consumed as fresh fruits or are dried to produce pepper powder. Those hot peppers can be divided into red hot pepper and sweet pepper based on their spiciness. While most other countries use the term “paprika” for chili or hot peppers, in Korea, “paprika” is used interchangeably with bell pepper to refer to the large sweet peppers in a variety of colors such as green, orange, yellow, and red. The production of bell peppers, mostly consumed as fresh fruit, has been increasing in Korea. According to the Korea Seed and Variety Service registry, approximately 600 hot pepper and 60 bell pepper cultivars have been developed to date by more than 50 seedling companies. Most pepper cultivars grown in Korea are of the red hot pepper variety, used to produce red hot pepper powders, one of the most important spices in Korean cuisine, used in kimchi, pastes, and other side dishes .Pepper plants are usually cultivated from April to October in Korea, and red hot pepper fruits are harvested from July to October. Diseases, such as phytophthora blight and anthracnose, and harmful insects, such as whitefly, oriental tobacco budworm, and thrip, cause serious damage to the quality and quantity of pepper production. Therefore, a variety of pesticides are regularly sprayed on pepper plants. However, excessive pesticide use poses a major risk to human and environmental health.Geminivirus and RNA viruses with single- or double-stranded RNA genomes are major viruses that infect pepper plants [In addition to harmful bacteria, fungi, and insects, several viruses are also known to infect pepper plants. For example, DNA viruses belonging to the genus r plants . Viroidsr plants .In Korea, the most frequently identified viruses infecting pepper plants are the cucumber mosaic virus (CMV), broad bean wilt virus 2 (BBWV2), and tomato spotted wilt virus (TSWV) . Hot pepWhile some farmers grow pepper seedlings, most purchase them from different nurseries. In many cases, nurseries cultivate multiple plant varieties within a limited space, such as in a greenhouse, potentially increasing the frequency of viral infections. In this study, we examined the viromes of 15 different pepper cultivars derived from 5 major nurseries in Korea, via RNA sequencing, followed by intensive bioinformatic analyses.We collected 15 pepper cultivars from 5 nurseries in Korea . The nurAll the bell pepper plants showed severe viral disease symptoms such as stunting, mosaic pattern, and curling . These pde novo transcriptome assembly using the Trinity program, we obtained 15 pepper transcriptomes. Each transcriptome was subjected to a BLASTX against the viral protein database. The identified virus-associated contigs were further subjected to a BLASTX against non-redundant protein databases to filter out non-viral sequences. Finally, 1325 virus-associated contigs were obtained. P14 contained the highest (213), and P01 showed the lowest (20) number of virus-associated contigs  were identified in this study and known to have multipartite genomes. For example, BBWV2, PCV2, and PCV1 had two RNA segments, whereas CMV had three. In total, 663 virus-associated contigs were derived from three CMV RNA segments: RNA1 (149 contigs), RNA2 (216 contigs), and RNA3 (298 contigs) B. A totaAll the examined pepper cultivars were coinfected with at least two viruses C. The nuWe also examined the number of pepper cultivars infected with the individual virus D. BBWV2 The proportion of viral reads in each transcriptome ranged from 0.04% (P06) to 24.5% (P04) A. The prde novo transcriptome assembly from 15 samples  were identified from the same pepper sample B,C. In eSimilarly, we identified 36 CMV genome sequences, CMV RNA1 (9 sequences), CMV RNA2 (6 sequences), and CMV RNA3 (21 sequences) C. IntereBased on the obtained viral genomes, we analyzed the phylogenetic relationships of the identified viral variants with known isolates for each virus. For BWYV, only a single variant, P03, was identified in this study. This variant showed sequence similarity to known BWYV isolates S14 and S19, which were identified in spinach in Japan. The phylogenetic tree using 11 BWYV isolates showed that BWYV variant P03 belonged to group A, possessing nine isolates of BWYV, whereas group B contained two BWYV isolates from France and the United States A.Although we identified two alphaendornaviruses, HPEV and BPEV, only two complete genomes of the BPEV variants, P14 and P15, were obtained. The phylogenetic tree using 24 BPEV isolates demonstrated that P14 and P15 belonged to group A containing 18 BPEV isolates B. Most oWe identified a single PCV1 variant in the P12 library. The PCV1 genome consists of two RNA segments: RNA1 and RNA2. The phylogenetic tree based on the 13 PCV1 RNA1 sequences revealed two groups of PCV1 isolates C. RNA1 oFor PCV2, composed of two RNA segments, we obtained three and two complete genome sequences for RNA1 and RNA2, respectively. We generated two phylogenetic trees based on the 21 PCV2 RNA1 and 20 PCV2 RNA2 sequences E,F. BothThe genomes of four PVY variants were obtained in this study. According to a previous study, several strains of PVY have been identified . We inclFor BBWV2, which is composed of two RNA segments, two different phylogenetic trees were constructed based on the individual RNA segments obtained in this study B,C. The We obtained 15, 13, and 21 sequences for CMV RNA1, RNA2, and RNA3, respectively. We constructed three phylogenetic trees based on individual RNA segments D–F, whicC. annuum (Ca18SrRNA) was used as a positive housekeeping gene for RT-PCR [To confirm the results of RNA sequencing, we conducted RT-PCR using virus-specific primers. To detect viruses infecting pepper plants, we designed different primer pairs for nine viruses . A primer RT-PCR . The desViruses and viroids that infect pepper plants have been intensively examined in many countries ,6,8. HowWe found that the most prominent viruses infecting pepper plants in Korea were BBWV2 and CMV. Both RNA sequencing and RT-PCR results revealed that all 15 cultivars were infected by BBWV2, while 13 cultivars were infected by CMV. Infection of BBWV2 has been widely reported in cultivated pepper plants in Korea . We assuAlphaendornavirus in the family Endornaviridae [In addition to hot pepper cultivars, we examined viruses infecting four bell pepper cultivars: P02, P09, P14, and P15. As expected, BPEV was identified in all the bell pepper cultivars. To our knowledge, this is the first study to report BPEV infection in bell pepper plants in Korea. In addition, we identified BPEV in a single hot pepper . Infection of hot peppers with BPEV has not been previously reported. This is the first study to report BPEV infection in hot pepper cultivars. HPEV was first identified in hot peppers in Korea . Interesaviridae . Their gAccording to a previous study in 2005, the most common viruses infecting pepper plants in Korea were CMV 19.8%) followed by PepMoV (13.4%), PMMoV (1.1%), BBWV2 (3.8%), and TMGMV (0.5%) . Some vi% followePVY was initially identified in bell peppers (paprika) in Korea . In our Due to differences in pepper samples and collection regions, other known viruses infecting pepper plants, such as PepMoV, PMMoV, and TMGMV, were not detected in this study. Moreover, in contrast to other Asian countries such as India  and VietIn contrast to most previous studies that have used RT-PCR and ELISA assays to identify viruses infecting pepper plants in Korea , we usedIt is noteworthy that the small number of reads derived from RNA sequencing might have been caused by cross-contamination from another library. For example, three reads for CMV RNA3 in the P01 cultivar and one read each for CMV RNA2 and CMV RNA3 could be cross-contaminants. The RT-PCR results demonstrated that CMV was not present in the P01 and P07 cultivars.Phytophthora resistance (PR). In addition, we included several pepper cultivars that were resistant to anthracnose and viruses. For example, TS-Monster (P03) was developed for resistance against TSWV. Chegangtan (P06) is resistant to anthracnose. Caltan (P12) is resistant to CMV, TSWV, and anthracnose. Interestingly, most disease-resistant pepper cultivars showed no viral disease symptoms, despite being coinfected with several viruses.Due to the coinfection of different viruses in each cultivar, it was difficult to determine the major virus-causing disease symptoms. However, the proportion of viral reads in each library made it possible to detect the virus responsible for the disease symptoms in each cultivar. For example, while HPEV was the dominant virus in the P01 cultivar coinfected with HPEV and BBWV2, P01 (PR-9988 cultivar) did not display any disease symptoms. The viral abundance of BBWV2 was very low in P01. Recently, several pepper cultivars resistant to diseases such as phytophthora blight have been developed in Korea, and the disease-resistant cultivar is referred to as Of the examined pepper cultivars, all four bell pepper cultivars displayed severe disease symptoms accompanied by improper growth and fruit production. To identify the major viruses causing disease symptoms in each bell pepper cultivar, we examined the dominant viruses based on the proportion of the viral reads. In P02, PCV2 was the most prominent virus, while CMV was the dominant virus in P09, P14, and P15. Based on this result, it was inferred that CMV may play a major role in the development of viral diseases in bell pepper plants. However, it was unclear whether PCV2 was the main virus causing disease symptoms in P02 because the PCV2-mediated disease symptoms have not been revealed to date. Moreover, BBWV2 and CMV may have coinfected the P02 cultivar. Although bell peppers are currently regarded as important horticultural plants in Korea, the viruses infecting bell peppers have not been intensively studied. In this study, we provided detailed information on viruses that infect major bell pepper cultivars.BBWV2 is currently regarded as an important virus causing severe damage to pepper production in Korea. As expected, our results showed that BBWV2 was the dominant virus in 7 out of 15 cultivars: P03, P06, P07, P08, P10, P12, and P13. Therefore, it is important to prevent the spread of BBWV2 in pepper fields. Both CMV and BBWV2 can be transmitted by aphids and mechanically via sap. In addition, CMV is transmitted by seeds ; howeverViral quasispecies is defined as population structure containing a large number of variant genomes caused by mutations . In our In addition, other assembled virus genomes such as BWYV, BPEV, PCV1, and PCV2 obtained in this study were successfully employed for phylogenetic analyses to reveal genetic relationships with other known viral genomes.The pepper plants used in this study originated from five different nurseries. Therefore, it is possible to track the origins of the identified viruses. However, we did not find any correlation between viral infections and the selected nurseries. Rather, most pepper cultivars displayed a cultivar-specific virome.Seedlings of 15 pepper cultivars were obtained from 5 nurseries . The seeFrozen pepper leaf samples were ground using a mortar and pestle in liquid nitrogen. The ground powder (100 mg) was subjected to total RNA extraction using an RNeasy Plant Mini Kit , according to the manufacturer’s instructions. The quality of the extracted total RNAs was measured using a Technologies 2100 Bioanalyzer . The total RNAs with RNA Integrity Number (RIN) values greater than or equal to 7 were used for library preparation. The libraries for RNA sequencing were prepared by depleting ribosomal RNAs from the total RNAs using TruSeq Stranded Total RNA with a Ribo-Zero Plant kit , according to the manufacturer’s instructions. A total of 15 libraries representing 15 pepper cultivars were prepared and, referred to as P01–P15.de novo transcriptome assembly using the Trinity assembler (version 2.13.2) with default parameters [The indexed libraries were paired-end-sequenced (150 bp × 2) using the Illumina HiSeq X system (Illumina). The raw RNA sequencing data were deposited in the sequence read archive (SRA) database in the National Center for Biotechnology Information (NCBI) with the respective accession numbers . The rawrameters .The assembled contigs from each library were subjected to a BLASTX search against the complete viral reference protein database derived from the NCBI using the DIAMOND program with an E-value of 1E-5 as cutoff . BLASTX Viral sequences covering all ORFs for each virus species were selected. The sequences were subjected to the NCBI ORFfinder . IndividThe assembled viral genomes for each virus species were subjected to BLASTN against the NCBI nucleotide database. The known viral genome sequences for each of the identified virus species were extracted. The viral genome sequences from this study and the known viral genome sequences from GenBank were aligned using MAFFT version 7 with the auto option . The aliPrimers were designed for recombinase amplification assays using the PrimedRPA program, to detect a total of nine viruses, namely CMV, BBWV2, BWYV, BPEV, HPEV, PCV1, PMMoV, PepMoV, and TSWV . RT-PCR RT-PCR was performed using a SoGent DiaStar One-Step RT-PCR Kit . The PCR reaction contained the following elements: 1 µL of template RNA (100 ng), 1 µL of the forward primer (10 pmol/µL), 1 µL of the reverse primer (10 pmol/µL), 6 µL of 5× Band Doctor, 6 µL of 5× buffer and 2 µL of the enzyme mix adjusted to a final volume of 30 µL using RNase-free water. RT-PCR was carried out using the following conditions: cDNA synthesis at 50 °C for 30 min, initial denaturation at 95 °C for 15 min, denaturation at 95 °C for 20 s, annealing at 60 °C for 40 s, and extension at 72 °C for 40 s. The denaturation-to-extension steps were repeated 34 times, with a total of 35 cycles continued to a final extension at 72 °C for 5 min. The samples were maintained at 4 °C. The PCR products were separated via gel electrophoresis on 1.5% agarose gel in the presence of a TBE buffer. The PCR products were stained using ethidium bromide and visualized under ultraviolet light.In this study, we carried out a comprehensive virome study of 15 pepper cultivars derived from 5 nurseries based in Korea. All four bell pepper cultivars showed severe viral disease symptoms; however, most hot pepper cultivars were either asymptomatic or had mild viral disease symptoms. RNA sequencing followed by bioinformatic analyses identified 1325 virus-associated contigs that were derived from 8 different RNA viruses. The proportion of viral reads in most pepper cultivars was relatively high, ranging from 0.04% to 24.5%. We found that BBWV2 and CMV were the most frequently identified viruses, followed by PVY. BBWV2 was the dominant virus in seven cultivars, whereas CMV was the dominant virus in five cultivars. Moreover, we reported, for the first time, the infection of BPEV in four different bell pepper cultivars in Korea. Although the pepper plants in this study originated from different nurseries, most pepper cultivars displayed a cultivar-specific virome. A total of 111 complete viral sequences for the 7 viruses were obtained. Using the obtained viral genome sequences, we revealed the genetic relationship between the identified viral isolates and known isolates for each virus. Moreover, we revealed quasispecies of BBWV2 and CMV in several pepper cultivars. We developed RT-PCR primers specific for nine RNA viruses infecting pepper plants, confirming the RNA sequencing results. Taken together, this study revealed the viromes of 15 pepper cultivars through RNA sequencing.


Individuals with autism spectrum disorder (ASD) are more likely to use healthcare than their counterparts without disabilities, which imposes high medical costs to families and health systems. This study aimed to investigate healthcare costs and its determinants among individuals with ASD.In this systematic review, we searched online databases  for observational and experimental studies that included data on service use and costs associated with ASD and published between January 2000 and May 2021. Exclusion criteria included non-English language articles, duplicates, abstracts, qualitative studies, gray literature, and non-original papers .Our searches yielded 4015 articles screened according to PRISMA guidelines. Of 4015 studies identified, 37 articles from 10 countries were eligible for final inclusion. Therapeutic interventions, outpatient visits and medications constituted the largest proportion of direct medical expenditure on individuals with ASD. Included studies suggest lack of health insurance, having associated morbidities, more severe symptoms, younger age groups and lower socioeconomic status (SES) are associated with higher medical expenditure in individuals with ASD.This systematic review identified a range of factors, including lower SES and lack of health insurance, which are associated with higher healthcare costs in people with ASD. Our study supports the formulation of policy options to reduce financial risks in families of individuals with ASD in countries which do not have a tax-based or universal health coverage system. Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects social abilities, communication skills and behavioral patterns among people with ASD . This diIndividuals with ASD require behavioral interventions and specialty health services to improve social interactions, communication skills and daily functioning. Evidence suggests that individuals with ASD are more likely to have higher rates of utilization of both acute care  and specialty care (e.g. psychiatric and neurology visits) than their counterparts without ASD , 6. For Studies show a strong relationship between co-occurring conditions and healthcare cost among individuals with ASD. Of these, mental health problems have a major effect on medical care costs. Peacock et al. found that children with ASD and intellectual disability incurred expenditures 2.7 times higher than children with ASD and no co-occurring condition . SimilarThe economic burden associated with ASD has been investigated in various studies. Studies have reported higher healthcare costs among individuals with ASD compared to the general population. For example, a study by Lavelle et al. indicated that annual costs of healthcare among children with ASDs were more than four times higher  than those without ASDs . Higher Some factors, such as age, place of residence, the level of disability, and associated comorbidities, can intensify the economic burden carried by individuals with ASDs. For example, Knapp et al. in the UK, found that lifetime costs are substantially higher for individuals with both ASD and intellectual disability than for those with ASD alone (£1.23 million vs £0.80 million) . SimilarOverall, the consequences of ASD can potentially affect employment, family relationships, standards of living, social interactions, personal functioning, and quality of life for individuals and their families. In this systematic review, we aimed to identify factors associated with healthcare costs among individuals with ASD. Total costs associated with ASD can vary between individuals and between countries, as a result of a number of different factors. However, identification and quantification of the healthcare, and other, costs associated with ASD can provide a comprehensive picture of the financial impact on families and health systems and can help to identify factors affecting those costs, which may be amenable to change or policy intervention. To ensure appropriate planning of the afore-mentioned services and allocation of resources, it is essential to identify the major contributors to healthcare costs among individuals with ASD. Therefore, the present study aimed to identify determinants of healthcare costs among individuals with ASD.Our study was a systematic review that adheres to the PRISMA guidelines . Three oWe included observational  and experimental  studies. As shown in Table Individuals with ASDStudies reporting healthcare costs for individuals with ASDObservational studies  or experimental studies  Published in English between January 1, 2000 and May 31, 2021Full-text articlesPublished before January 1, 2000 and after May 31, 2021Qualitative research, case studies, and literature reviews (e.g. scoping and systematic reviews)Abstracts, editorials, letter to editor, and commentsProtocols and method papersGrey literature Non-English language studiesWe developed a data extraction form to extract information such as authors, year, country, study design, study sample, costs, study perspective , and paRegarding the design of the included studies, we applied JBI Critical Appraisal Checklists for cross sectional studies and cohort studies to assess the quality of the studies included in the review . QualityThe process of study selection is shown in Fig. n = 21), the United Kingdom ; both USA and UK (n = 1),Taiwan (n = 2), Ireland (n = 1), Germany (n = 1), Israel (n = 1), China (n = 1), Australia (n = 1), Oman (n = 1), and South Korea (n = 1). The age of participants ranged between 1 and 89 years and the majority of studies focused on ASD or ASD with and without intellectual disability.The 37 included studies, which are summarized in Table Included costs varied between studies, as illustrated in Table Outpatient and community-based services, such as physician appointments, were more commonly used than inpatient care and thus these services would impose a high economic burden on families of individuals with ASD in countries where such services are paid out of pocket. The study by Barrett et al. (2012), in the UK, reported a higher proportion of the included sample utilizing outpatient and community-based appointments, with 60, 57 and 48% of children with ASD having at least one contact with community pediatricians, general practitioners, and health visitors , respectively, over a 6 month period, compared to 10% of participants reporting an inpatient episode of care . SimilarThe included studies suggest that prescription drugs make a major contribution to expenditure for households. Mosadghrad et al., in Iran, for example, estimated the average annual direct medical costs of ASD? were approximately 2215 USD of which 16% consisted of medication, compared to 7 and 6.5% for outpatient and inpatient visits, respectively . SimilarEight studies (24.24% of included studies) reported that inpatient services, equipment and emergency room visits constituted a smaller proportion of total healthcare costs compared to outpatient services. For example, a study by Thomas et al. (2014), in the USA, showed that inpatient services constitute only 0.5% of expenditure compared to outpatient services (27.7%) . Young eIndirect costs were commonly estimated in studies involving children and young people and were focused on lost productivity for parents and carers. Three studies (9.09% of included studies) showed that indirect costs constitute a greater proportion of total costs (direct + indirect) compared to healthcare expenditures in families of individuals with ASD. Accordingly, a study by Al-Farsi et al., in Oman, indicated a higher monthly indirect cost  in contrast to direct healthcare expenditure  . In theiIn contrast, Mosadeghrad et al., carried out in Iran, reported mean annual indirect costs due to productivity loss of 1118 USD, which was a smaller contribution to total expenditure (16%) compared to direct medical costs (32%) and direct non-medical costs (52%) . In the Table In some included studies, associated morbidities were one of the significant contributors to total healthcare expenditures in children with ASD. Van Steensel et al., in the USA, estimated the cost of children with ASD and comorbid anxiety disorder were four times higher than anxiety disorder alone, and 27 times higher than typically developing children . SimilarA number of included studies showed that increasing symptom severity was associated with increased costs. Raz et al., in Israel, suggests that individuals with a clinically more severe condition have a higher likelihood of very high costs  . HoweverSome included studies found that families in receipt of public health insurance, such as Medicaid, were more likely to demonstrate lower out of pocket costs compared with those in receipt of private insurance. A study by Parish et al., in the USA, showed that the median annual out of pocket costs in low-income families of Medicaid–insured individuals with ASD was 7 USD, compared with 160 USD for low-income families with private insurance coverage .Studies also suggest that a lack of insurance coverage increases the probability of high out of pocket expenditures , 2. ThomSeven studies showed a positive association and three studies indicated a negative association between age and healthcare costs among individuals with ASD. In adults, Zerbo et al., in the USA, found that the annual heath care cost in adults aged 50 years and older  was higher than for the 18–25 age group (5000 USD) .In younger populations, some studies showed that younger age groups were associated with higher costs than older age groups. For example, Barrett et al. (2015), in the UK, found that for every one-year increase in age, total health, education and social care costs fell by £4917 among adolescents with autism and autism spectrum disorder aged between 14 and 17 . SimilarIncluded studies suggest that households with higher socioeconomic status (SES) were more likely to pay out of pocket for healthcare in comparison to those with lower SES. For instance, Raz et al., in Israel, and Thomas et al., in the USA, reported that higher parental education and higher household income were associated with higher out of pocket expenditure in families of children with ASD , 39. On This systematic review aimed to identify factors contributing to healthcare costs among individuals with ASD. Our findings showed that therapy services, outpatient visits, and medications made the largest contributions to total direct healthcare costs of ASD compared to inpatient care in the included studies, suggesting ASD is primarily managed in the community. Studies show that individuals with ASD are more likely to use therapy services  than those without ASD which in turn imposes high costs on families and health systems , 25.Insurance coverage was an important factor influencing the use of services like psychological and rehabilitation therapies, which are known to be important for improving competencies and facilitating social and economic participation in individuals with intellectual disabilities . For exaIn countries with public insurance, such as the USA, included studies suggest that -children in receipt of public insurance had a lower out of pocket financial burden compared to uninsured and privately insured children . Our revIncluded studies showed that outpatient visits and medications constituted a major share of direct medical costs among individuals with ASD , 25, 29.The impact of age on healthcare costs was conflicting, with some suggesting a positive association and others suggesting a negative association. For adults, older age was associated with higher costs. However, the opposite was true in some studies focused on children and young people, with evidence that younger ages are associated with higher physician visits, prescription medication and related costs among individuals with ASD , 40, 44.Whilst the focus of the current paper was on healthcare costs, some included studies took a broader perspective and investigated societal costs among individuals with ASD , 37, 50.A number of included studies estimated indirect costs, in addition to direct healthcare costs, and these studies suggest that indirect costs constitute a major part of the total cost of individuals with ASD , 24, 33.Our study faced some limitations. Firstly, we were not able to compare total healthcare costs and out of pocket expenses between included studies because of heterogeneity in the cost components included in each study. Second, healthcare delivery and financing schemes varied across the included studies, making it difficult to compare healthcare costs between countries. Third, we did not include grey literature and non-English language articles which may have affected the findings and the generalizability of the present study. Finally, we only included studies that reported individual costs per person; aggregate medical and non-medical costs for individuals with ASD should be investigated at local and/or national levels in future studies.This systematic review showed that therapy services, outpatient visits and medications constituted a major share of total direct healthcare costs among individuals with ASD. Indirect costs were also considerable, being higher than direct healthcare costs in some studies. Key determinants of healthcare costs in the included studies were health insurance, associated morbidities, symptom severity, age and socioeconomic status. Our study supports the formulation of policy options to reduce financial risks in families of individuals with ASD.


Plantago asiatica mosaic virus belongs to the genus Potexvirus in the family Alphaflexiviridae of the order Tymovirales.Plantago asiatica mosaic virus (PlAMV) has flexuous virions of approximately 490–530 nm in length and 10–15 nm in width. The genome of PlAMV consists of a single‐stranded, positive‐sense RNA of approximately 6.13 kb. It contains five open reading frames (ORFs 1–5), encoding a putative viral polymerase (RdRp), movement proteins , and coat protein (CP), respectively.Plantago asiatica, Nandina domestica, Rehmannia glutinosa, and other weed plants. Experimentally PlAMV can infect many plant species including Nicotiana benthamiana and Arabidopsis thaliana. It also infects ornamental lilies and frequently causes severe necrotic symptoms. However, host range varies depending on isolates, which show significant biological diversity within the species.PlAMV has an exceptionally wide host range and has been isolated from various wild plants, including PlAMV can be separated into five clades based on phylogenetic analyses; nucleotide identities are significantly low between isolates in the different clades.PlAMV is not reported to be transmitted by biological vectors. Virions of PlAMV are quite stable and it can be transmitted efficiently by mechanical contact.P. asiatica.PlAMV causes red‐rusted systemic necrosis in ornamental lilies, but it shows much weaker, if any, symptoms in wild plants such as Control of the disease caused by PlAMV is based mainly on rapid diagnosis and elimination of the infected bulbs or plants. This pathogen profile summarizes current knowledge regarding Plantago asiatica mosaic virus, including its host range, strain differentiation, host interactions, and utility as a viral vector to examine virus–host interactions. Here we summarize current knowledge regarding PlAMV, including its natural and experimental host range, strain differentiation, host interactions, and utility as a plant viral vector to examine virus–host interactions.22.1P. asiatica, a perennial herbaceous species endemic to north‐eastern Asia, by Kostin and Volkov  in California, USA  in Japan , and Stellaria media, Primula vulgaris, and Urtica urens in the Netherlands  and Pelargonium inquinans (LC667834) in Korea, and Epimedium sp. (MZ344590) in Canada. The natural hosts are shown in bold in Table Further isolates were found in other plant species and countries, including 2.2P. asiatica, a partial experimental host range was determined, including 24 species from 12 plant families . Several experimental hosts listed in Table When PlAMV was first discovered in 2.3No biotic vector of PlAMV is known. As with other potexviruses, PlAMV is readily transmitted by mechanical inoculation with sap extracts Conijn, , but als2.4P. asiatica symptoms are mottling, mottled chlorosis  were obtained from a single infected N. benthamiana without single lesion passaging. Each showed varying symptoms within and between single plants of N. benthamiana, typically chlorotic to necrotic local lesions, followed by systemic mottle or mosaic. Some leaves with mosaic developed necrotic patches, often spreading down the veins into the petiole and the main stem; other upper leaves developed narrow areas of white tissue surrounding areas of mosaic (authors' unpublished data). This suggests the presence of a mixture of sequence variants that compete for predominance within the same plant, similar to the occurrence of Li1–Li6 in L. leichtlinii or variants of Alternanthera mosaic virus (AltMV) in Phlox stolonifera .Naturally infected g Figure , while tg Figure . PlAMV iNicotiana species, notably N. benthamiana, N. edwardsonii, N. megalosiphon, and N. occidentalis, developed clear systemic mosaic that frequently became necrotic in a significant proportion of the leaves, with symptom severity varying both within and between individual plants  are highly genetically homogenous, suggesting a common origin of these isolates , stem‐loop structures were identified in both UTRs by RNA folding predictions, which may be important for replication. Indeed, a PlAMV “replicon”, which consists of only the replicase ORF flanked by 5′‐ and 3′‐UTRs, can produce minus‐strand genomic RNA  peptide ‐expressing vector of PlAMV, widely used as a model virus infecting 4.2ORF1 encodes a replicase, which contains three conserved domains: a methyltransferase, a helicase, and an RNA‐dependent RNA polymerase Figure . ReplicaORF2, ORF3, and ORF4 encode TGBps required for cell‐to‐cell movement. Indeed, disruption of each of these TGBps by mutation of their initiation codon inhibits cell‐to‐cell movement of PlAMV  proteins and RNA silencing  and Suppressor of Gene Silencing 3 (SGS3), host antiviral factors required for the trans‐acting small interfering RNA synthesis pathway ‐promoter assay showed JAX1 to be highly expressed in the vascular tissue, completely inhibiting systemic infection of potexviruses. This vascular‐specific expression and complete inhibition of systemic viral infection resembles that of the jacalin‐type lectin gene RTM1 of A. thaliana, which confers resistance against tobacco etch virus, but the spectrum of resistance of JAX1 and RTM1 was different; JAX1 confers resistance against potexviruses in general, while RTM1 is effective against potyviruses  is a dominant resistance factor that restricts PlAMV at the single‐cell level  is the first identified recessive resistance gene against PlAMV infection and inhibits replication at the single‐cell level  was reported to restrict PCD during bacterial and oomycete infections , a key regulator of defence gene expression in the salicylic acid pathway. A plant immune activator, acibenzolar S‐methyl (ASM), restricted PlAMV infection at a single‐cell level, which requires NPR1  detection of PlAMV, using either generic potexvirus primers  has also been used and is more suitable for quantifying PlAMV titre than other assays. Tanaka et al.  developeA reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) assay was developed , pesticide applications are unlikely to control its spread.Many countries have imposed strict standards for testing lily bulbs for import or export. Meristem tip culture, especially when combined with thermotherapy and/or chemotherapy, can result in recovering virus‐free plants. However, the plant material in tissue culture for international distribution or micropropagation should also be subjected to rigorous testing and selection. It is known that tissue culture itself can sometimes result in reduction of virus titre below the sensitivity of normal RT‐PCR detection. An initially undetectable virus titre can slowly build up over a number of weeks after acclimation of tissue‐cultured material to the greenhouse, therefore retesting plants after several weeks in the greenhouse is needed to select founder material for a nuclear stock.PlAMV infection can be transferred to previously healthy lily bulbs during the washing and processing that occurs after bulb harvest  or dicots . Second, PlAMV shows multiple virus–host interactions involving various virus‐encoded proteins and five clades of diverse isolates spanning an array of natural host species. These should allow PlAMV gene exchanges to determine the factors affecting host range and symptom severity. Finally, a GFP‐labelled infectious clone was successfully developed to examine both similarities and differences of host interactions affecting levels of viral replication, cell‐to‐cell movement, and long‐distance movement in the same plant host.The extent of the work on PlAMV that has resulted from this interest has revealed several features that make PlAMV an attractive model system to complement knowledge obtained from other well‐studied viruses in the genus The features of PlAMV summarized in this review therefore recommend PlAMV as a highly flexible model virus system, with an established knowledge base, suitable for addressing many questions in a wide variety of host plants and permitting commonalities and differences between monocot and dicot host systems to be probed with a single model virus.The authors declare no conflict of interest.


Here, we used well-established mouse and porcine models to study infection phenotypes of the CpG-enriched neurotropic and congenital virus—Zika virus, directly in the target tissues—the brain and placenta. Specifically, we used the uttermost challenge and directly injected mice intracerebrally to compare infection phenotypes caused by wild-type and two CpG-recoded Zika variants and model the scenario where vaccine strains breach the blood-brain barrier. Also, we directly injected porcine fetuses to compare in utero infection phenotypes and model the scenario where recoded vaccine strains breach the placental barrier. While overall infection kinetics were comparable between wild-type and recoded virus variants, we found convergent phenotypical differences characterized by reduced pathology in the mouse brain and reduced replication of CpG-enriched variants in fetal lymph nodes. Next, using next-generation sequencing for the whole virus genome, we compared the stability of de novo introduced CpG dinucleotides during prolonged virus infection in the brain and placenta. Most de novo introduced CpG dinucleotides were preserved in sequences of recoded Zika viruses showing the stability of vaccine variants. Altogether, our study emphasized further directions to fine-tune the CpG recoding vaccine approach for better safety and can inform future immunization strategies.Increasing the number of CpG dinucleotides in RNA viral genomes, while preserving the original amino acid composition, leads to impaired infection which does not cause disease. Beneficially, impaired infection evokes antiviral host immune responses providing a cutting-edge vaccine approach. For example, we previously showed that CpG-enriched Zika virus variants cause attenuated infection phenotypes and protect against lethal challenge in mice. While CpG recoding is an emerging and promising vaccine approach, little is known about infection phenotypes caused by recoded viruses  Targ in vivo –9 providhallenge . Our CpGhallenge .in vivo. Attenuated infection phenotypes caused by recoded vaccine candidates may depend on the expression of cellular components targeting viral CpG dinucleotides , amplified with high fidelity PCR (Invitrogen) and transfected into C6/36 Aedes albopictus mosquito cells (ATCC #CRL-1660) at +37°C for 12 h, and incubated for 7 days at +28°C (de novo introduced CpG dinucleotides after ten passages in VERO cells (® Mycoplasma PCR Detection Kit (Sigma).To recover Zika virus variants, we used Infectious Subgenomic Amplicons (ISA) –19 as prat +28°C . Media fRO cells . All vir3 TCID50 in 30 µl DPBS) as we and others previously described  biosafety level 2 mouse facility. To compare local brain-specific infection phenotypes caused by different Zika virus variants, BALB/c male and female mice  were inoculated intracerebrally (IC) with the virus   as previously described . The control pig was inoculated with virus-free media. Pregnant pigs were euthanized and sampled 28 days after in utero inoculation. Uteri with fetuses were removed. Amniotic fluid and uterine wall with the placenta  were collected from each conceptus and rapidly frozen. Amniotic fluid was aspirated with sterile syringes with needles before tissue dissection. All samplings were performed in the direction from non-manipulated fetuses closest to the right uterine horn tip toward inoculated fetuses. Fetuses were visually examined. Blood plasma was collected from all fetuses; after blood centrifugation , plasma was aliquoted and frozen (-80°C). Amniotic fluid, inguinal lymph node, thymus, spleen, and liver were sampled from all fetuses. Fetal brains were removed from all fetuses and frozen in liquid nitrogen. Body and brain weights of viable fetuses were measured. Selected fetal samples were tested by PCR for porcine circovirus 2 (PCV 2), porcine circovirus 3 (PCV 3), PRRSV and PPV  and porcine parvovirus (PPV) which can cause fetal infection. Pregnant pigs were randomly assigned into Control, E+102CpG, E/NS1+176CpG and WT groups. Results from the pig exposed to the only wild-type virus were partially reported in our previous study . All aniescribed –26. For  and PPV . All samThe lateral surface of the frozen right cerebral hemisphere in adult mice and pig fetuses were shaved with sterile scalpel blades to collect 20-40 mg of tissue. Tissue samples were kept on dry ice and weighed on analytical balances. One ml of TRI Reagent Solution (Invitrogen) was added, and tissues were homogenized using RNase-free stainless-steel beads and TissueLyser II (QIAGEN) operating for 5 min at 25 Hz. Then RNA extraction was performed with PhaseMaker tubes (Invitrogen) and PureLink RNA Mini Kit (Invitrogen) according to the manufacturer’s instructions. For porcine placenta, amniotic fluids, lymph nodes, thymus, spleen, liver and mouse spleen, PureLink RNA Mini Kit was used to extract RNA according to the manufacturer’s instructions.2−109 virus RNA copies/reaction for the limit of quantification) with a high linear correlation (R2 = 0.9997) between the cycle threshold (Cq) value and template concentration. The slope of the standard curve (-3.4351) corresponded to the 95.5% reaction efficiency level. All samples with PCR values below the limit of quantification (LOQ) had values below the limit of detection ; thus, only LOQ is represented in graphs. In all PCR tests, we used VERO cell culture media containing Zika virus as a positive PCR control. As a negative control, we used samples from mock-inoculated control animals. Strict precautions were taken to prevent PCR contamination. Aerosol-resistant filter pipette tips and disposable gloves were used. Kit reagent controls were included in every RNA extraction and PCR run.Zika virus-specific SYBRgreen-based one-step RT-qPCR  was cond50 of Zika virus and incubated for 2 h . Then 100 μl of the culture medium  was added and after incubation  plates were dried and stored at -20 °C until use. Plates were thawed and cells were fixed in 10% buffered formalin . Cells were washed twice with 1x DPBS (pH 7.2) and incubated with 100% methanol in the presence of 0.3% H2O2 . Then plates were washed with DPBS and two-fold serial dilutions of blood plasma were added, followed by 1h incubation (+37 °C). Plates were washed three times with DPBS containing 0.05% Tween-80 and 50 μl/well of rabbit anti-pig IgG  or goat anti-mouse IgG  antibodies conjugated with horseradish peroxidase were added. After incubation  and washing, color reaction was initiated by adding substrate solution , 10 mM H2O2). The reaction was stopped by replacing the substrate with an acetate buffer and Zika virus-specific staining was determined by examination with a microscope. The titers were defined as the log reciprocal of the highest serum dilution. Blood plasma from mock-inoculated control animals was used as a negative control.To quantify Zika virus-specific IgG antibodies in mouse and pig blood plasma, we used immunoperoxidase monolayer assay (IPMA) –26, 30. β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-17A (IL-17A), interferon gamma (IFN-γ), and interferon alpha (IFN-α) in blood plasma and amniotic fluid as previously described  staining to screen lesions.® II Reverse Transcriptase  and Random Hexamers . As previously validated for accurate SNV calling, we used more than 1,000 viral RNA copies for cDNA synthesis . An enzyme activation step of 30 s at +95°C was followed by 35 amplification cycles for 15 s at +95°C and 5 min at +65°C. The correct size of amplicons in each reaction was verified by agarose gel electrophoresis.RNA from virus stocks, brain, and placenta samples were used to construct Zika virus whole-genome NGS libraries; libraries for mouse brain and porcine placental samples were constructed independently. While we found Zika RNA in lymph nodes from 84.6% of porcine fetuses in the wild-type group and 42.8% of fetuses in the E/NS1+176CpG group, viral loads in E/NS1+176CpG fetuses were not sufficient to construct NGS libraries. Thus, we only used mouse brain and porcine placental samples for evolutionary analysis. We used the PrimalSeq protocol, an approach wherein a nearly whole virus genome is amplified in the ~400–480 bp overlapping fragments with multiplexed PCR reactions and with two technical replicates for each multiplex reaction . The ampynthesis . Zika viynthesis . To acco® Ultra™ II DNA Library Prep Kit for Illumina® (NEB) and NEBNext® Multiplex Oligos for Illumina® (96 Index Primers) (NEB) according to the manufacturer’s protocol. Individual sample libraries were quantified with the Qubit dsDNA HR Assay Kit and Qubit 3.0 Fluorometer. The expected peak DNA fragment size (~580 bp) was confirmed with the Agilent DNA 1000 kit on Agilent 2100 Bioanalyzer (Agilent). In total, 20 ng of each barcoded library was pooled, quantified (Qubit 3.0), quality-checked , and converted to moles: Molecular weight [nM] = Library concentration [ng/µL]/((Average library size x 650)/1,000,000). The pooled library was diluted to 2 nM in 10 mM TE, denatured with 0.1 N NaOH and diluted to 14 pM, and paired-end 300 nt reads were generated with MiSeq Reagent kit v3 600 (Illumina) on the MiSeq System (Illumina). Access to sequencing data files generated in this study is available in the Sequence Read Archive (SRA) database under BioProject ID PRJNA837685.We constructed libraries and made Illumina sequencing as previously described with some modifications . Virus ain vivo samples, we used ancestral wild-type, E+102CpG, and E/NS1+176CpG sequences which were used to synthetically generate Zika virus variants .We used an open-source software package iVar  to process virus sequencing data and call single nucleotide variations (SNVs) . To idenAs per the previously described and validated best practices , we analH-test and Dunn’s multiplicity-adjusted post-test. The bodyweight dynamics were compared using a mixed-effects model. In the porcine model study, the number of dead fetuses was compared with the Yates-corrected χ2-test. Brain to body weight ratio, Zika virus loads in tissues and cytokine levels in the porcine fetuses were compared with the Kruskal-Wallis H-test and Dunn’s multiplicity-adjusted post-test. The correlation of IFN-a and IFN-γ levels in fetal body fluids with Zika virus loads in amniotic fluids was assessed with the Spearman correlation test. Genomic complexity between sequences from mouse brains and porcine fetuses inoculated with different Zika virus variants was compared with the Kruskal-Wallis H-test.We used GraphPad PRISM 8 software. Results with a statistically significant difference had p < 0.05. All data were expressed as individual values, mean, or median. The groups inoculated with different Zika virus variants were compared with the negative control group and between each other. In the mouse study, the Zika virus loads in tissues and antibody titers were compared with two-way ANOVA and Dunnett’s multiple comparisons test as well as with the Kruskal-Wallis 3 TCID50 of wild-type, E+102CpG, or E/NS1+176CpG Zika virus variants. Mice did not show changes in behavior and appearance. The body weight did not differ significantly between groups  average sequencing depth (de novo synthetize DNA fragments to rescuer variants) let us identify Zika virus stock- and variant-specific SNVs  could also contribute to the discrepancy. In support, in our ongoing unpublished study, synthetically-derived wild-type PRVABC59 variants, which have lower complexity than the patient-derived parental PRVABC59 strain, also do not cause fetal brain infection. In contrast, the patient-derived parental PRVABC59 strain causes infection in fetal brains.We did not detect Zika variants in the fetal thymus, spleen, and liver tissues. Brains in all fetuses in all groups were also negative for the virus. In all our previous studies with the patient-derived wild Asian PRVABC59 strain [GenBank: KU501215.1], Zika virus caused infection in fetal brains , 26. In  baboons . In addiIn utero infection kinetics were also similar: Regression analysis of variant loads in the placenta and amniotic fluid from directly inoculated and trans-infected fetuses showed similar efficiency of virus transmission between siblings within the uterus  average sequencing depth . Placental samples infected with the wild-type and E/NS1+176CpG variants did not show stock-specific sequences  and codon pair deoptimized (CPD) viruses have been studied in mice , 45. AllThe traditional rationale of CpG dinucleotide suppression in vertebrates by the methylation-deamination-mutation hypothesis cannot explain why RNA viruses also have an underrepresentation of genomic CpG dinucleotides . Althougde novo introduced CpG dinucleotides than the E/NS1+176CpG variant—did not cause histopathology in mouse brains. While E/NS1+176CpG caused histopathology comparable to the wild-type variant  repository, BioProject accession number PRJNA837685.via cardiac puncture. To ensure death, the chest cavity was opened during tissue collection. Pigs were euthanized with an anesthetic overdose and exsanguination.We conducted animal experiments following the Canadian Council on Animal Care standards for the ethical use and care of animals in science. Animal studies were approved by the University of Saskatchewan’s Animal Research Ethics Board (#20180012). All efforts were made to minimize animal suffering. Mice were euthanized by isoflurane and exsanguination Conceptualization, project administration, and supervision: UK. Data curation: DU and UK. Formal analysis and validation: DU, IT, and UK. Funding acquisition: DU and UK. Resources: UK. Investigation: DU, IT, NB, and UK. Methodology, software, visualization, and writing—original draft preparation: DU, IT, and UK. Writing—review and editing: DU, IT, NB, and UK. All authors contributed to the article and approved the submitted version.This work was supported by grants #420249 and #421645 to UK from Natural Sciences and Engineering Research Council (NSERC). DU received a Scholarship from the School of Public Health, University of Saskatchewan. VIDO receives operational funding from the Government of Saskatchewan through Innovation Saskatchewan and the Ministry of Agriculture and from the Canada Foundation for Innovation through the Major Science Initiatives for its CL3 facility (InterVac). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.We thank VIDO animal care technicians and veterinarians for help with animal experiments. Published as VIDO manuscript #970.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 efficiency with which ribosomes shift reading frames when decoding viral RNA may change over the course of an infection. Related research article Cook GM, Brown K, Shang P, Li Y, Soday L, Dinan AM, Tumescheit C, Mockett APA, Fang Y, Firth AE, Brierley I. 2022. Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression. eLife11:e75668. doi: 10.7554/eLife.75668.When an RNA virus infects a cell, ribosomes inside the cell decode the genetic information in the virus’s RNA to produce proteins, which are then used to make more viral particles. A single-stranded RNA molecule consists of a sequence of nucleotides that the ribosome reads three at a time. Each triplet, or codon, codes for either an amino acid (the building blocks that form proteins), or signals for the ribosome to start or stop reading the RNA sequence. Therefore, each nucleotide sequence can therefore be ‘read’ by ribosomes in three different ways, or ‘reading frames’, depending on which nucleotide the ribosome starts reading from. Additionally, an ‘open reading frame’ or ORF is a sequence of nucleotide triplets that code for amino acids located between two stop codons in the same reading frame.Almost all cellular proteins are encoded in a single reading frame, with only rare exceptions . VirusesIt is unclear exactly why viruses employ programmed ribosomal frameshifting. One suggestion is that this mechanism allows for a more compact organization of genetic material. Another is that frameshifting could be used for setting a specific ratio between different viral proteins. Most commonly, ribosomal frameshifting occurs during the synthesis of viral polyproteins, long amino acid chains that are processed into smaller proteins with distinct functions. The advantage of organizing protein synthesis in this way is that only one RNA molecule is needed to encode multiple proteins. However, if all these proteins were synthesized as a part of a single polyprotein, they would occur strictly in a one-to-one ratio after being processed. This would be wasteful, since these proteins are needed in different quantities.ORF1A, while proteins that the virus requires in lower quantities are encoded in a different but overlapping downstream reading frame, herein referred to as ORF1B  studied how viral gene expression changes during porcine reproductive and respiratory syndrome virus (PRRSV) infection. To do this, Cook et al. used a technique called ribosome profiling to map which parts of the virus’s RNA sequence were being translated by ribosomes at any given time . These mORF1A is higher than the footprint density at ORF1B. This happens because only a small proportion of the ribosomes reading ORF1A shift reading frame and proceed to ORF1B (Ribosome profiling can also be used to compare how efficiently different proteins are synthesized. For example, in the PRRSV genome the density of footprints mapped to to ORF1B . By calcORF1A and ORF1B, which is used by many viruses, and a second, rarer frameshift site in ORF1A that results in the production of a shorter polyprotein. The genome of a related virus, called the encephalomyocarditis virus, has been shown to have a similar secondary frameshift site that is stimulated by a viral protein (ORF1A and ORF1B.The PRRSV genome is known to contain two frameshift sites: the canonical site between  protein . The conORF1A and ORF1B as a mechanism that enables a fixed ratio between polyprotein products. The temporal change detected in PRRSV suggests that the efficiency of frameshifting may also be altered in other viruses over time. If so, it would be interesting to determine what factors mediate the regulation of the frameshifting event between ORF1A and ORF1B.This finding challenges the current paradigm that regards the canonical frameshifting between An open question that remains is how changes in frameshifting efficiency along the course of an infection relate to the virus’s virulence and transmissibility. It is possible that changes in efficiency are simply due to alterations in the infected cell that make ribosomes more prone to shifting to another reading frame. However, it may be that regulating the efficiency of frameshifting is beneficial for the virus. Alternatively, the antiviral response of the host may induce frameshifting to alter the ratio of viral proteins and negatively impact the virus. Indeed, it has been previously reported that the formation of viral particles can be disrupted by altering frameshifting efficiency .Whatever the case, the search for cellular factors responsible for changes in frameshifting has already begun . The ide


Most causes of boda-boda accidents and injuries were related to the behaviors of drivers, passengers, and pedestrians. The purpose of this study was to determine factors associated with boda-boda drivers, accidents, and boda-boda accident victims in Gulu Municipality, Northern Uganda.two cross-sectional studies were conducted at intervals of six months between July and December 2015. Two hundred boda-boda drivers from Gulu Municipality and fifty-seven victims of boda-boda accidents admitted to the Gulu Regional Referral Hospital were recruited for this study. A pre-tested questionnaire with Cronbach´s alpha  α=072 was used for data collection. This study was approved by a local Institutional Review Board (IRB) and STATA version 14.1 was used for statistical analysis. A p-value less than 0.05 was considered statistically significant.factors associated with boda-boda accidents in Gulu Municipality were boda-boda drivers from Pece division  and those with low monthly incomes less than UGX400,000/= equal to USD$100 . Drivers with monthly incomes higher than UGX400,000/= were least likely involved in boda-boda accidents . Work experience, prior road safety training, age, wearing a helmet and protective clothing, levels of education, and knowledge on road safety regulations did not significantly affect the outcome. Most victims of boda-boda accidents were passengers and pedestrians from villages outside Gulu Municipality  and sustained minor injuries.boda-boda accidents in Gulu Municipality are problematic, drivers from the Pece division and those with low monthly incomes were more involved. Most victims of boda-boda accidents were passengers and pedestrians from villages outside Gulu Municipality. This study suggests that boda-boda drivers should be engaged in other income-generating activities, as some divisions in Gulu Municipality do not generate the required resources to meet their needs. Boda-boda as described in the English Oxford Dictionary is a type of commercial motorcycle or bicycle with a space for a passenger or carrying goods, and often used as a commercial motorcycle . Its namCaptivatingly, the number of road traffic accidents attributable to boda-boda drivers has been increasing annually in Uganda and particularly in the urban areas . This waStudy design: two cross-sectional studies were conducted at six months´ intervals on two hundred boda-boda drivers in Gulu Municipality between July and December 2015. In the same period, fifty-seven victims of boda-boda accidents were recruited at Gulu Regional Referral Hospital.Study settings: this study was conducted in Gulu Municipality and Gulu Regional Referral Hospital. Gulu Municipality is one of the major towns in Northern Uganda, approximately 343 kilometers north of Kampala, the capital city of Uganda. It is reported that it is one of the fastest-growing urban centers in Uganda. It is also a major urban center in Northern Uganda and a gateway to lucrative businesses in South Sudan. The municipality is situated on the Great North Road which connects Cape Town in South Africa to Cairo in Egypt (North Africa). Data collection was undertaken at the boda-boda road-side stages located along major routes in the four divisions of Gulu Municipality . Each boda-boda stage was strategically located and was well organized with a management structure and a stage leader. The function of the leader was to regulate the operations of members and organize collective actions in times of need. On the other hand, Gulu Regional Referral Hospital is the main public hospital and a teaching hospital for Gulu University Medical School. It is located at the center of Gulu Municipality and offers free emergency medical and surgical services to victims of accidents. The hospital has an accident and emergency department (entry point), surgical wards , and operating theatres for the management of trauma cases. Hospital data for this study were collected from the surgical units of Gulu Regional Referral Hospital.Study population: the study population was boda-boda drivers from the four divisions of Gulu Municipality and victims of boda-boda accidents who were treated at the surgical units of Gulu Regional Referral Hospital .Selection of participants: boda-boda drivers were selected consecutively from boda-boda stages scattered along major routes in the four divisions of Gulu Municipality. Similarly, victims of boda-boda accidents were consecutively selected from the Surgical Units of Gulu Regional Referral Hospital from July to December 2015.Inclusion and exclusion criteria for drivers: for boda-boda drivers, three conditions had to be fulfilled before recruitment; 1) the driver was a registered member of a stage; 2) had been operating in Gulu Municipality for three or more months consecutively before this study; 3) had written informed consent. Those without the three requirements were excluded from the study.Inclusion and exclusion criteria for victims of boda-boda accidents: victims of the boda-boda accidents were recruited on condition that they had; i) a medical form five from Gulu Regional Referral Hospital with evidence that they had sustained boda-boda injuries in Gulu Municipality; ii) police confirmation; iii) written informed consent, and; (iv) the victim was conscious and capable of taking part in interviews. Those without the four requirements for recruitment were excluded from the study.A sampling of participants: boda-boda drivers and victims of boda-boda accidents were sampled consecutively from their work stations and Gulu Regional Referral Hospital Surgical Units respectively. We consecutively recruited drivers  to the study until the required sample size was attained. This method of sample size determination had been previously used in several studies conducted on boda-boda drivers in Uganda [n Uganda ,3,5,21. Sample size estimation: we used the total number of registered boda-boda drivers in Gulu Municipality as the total population for computing the sample size for our study population. Municipal authorities and the police reported six hundred (600) as the number of registered boda-boda drivers in Gulu Municipality [cipality . A previcipality ,21. We cWhere:Zα/2 is the critical value of normal distribution at α/2 ; MOE = the margin of error; p = the sample proportion; N = the total population size; n = sample population (sample size). The total population of registered boda-boda members was 600, with a 95% confidence level (1.96), an estimated prevalence of boda-boda in the community of 24.1% (0.241), and a margin of error of 0.05. We used 24.1% for calculating boda-boda accidents based on a previous study conducted in Gulu Regional Referral Hospital on boda-boda accidents [Considering a non-response rate of 5%, n=191.628 + 191.628 x 0.05 = 191.628 + 9.5814 = 201.2094. Therefore, the sample size obtained for this study population was 201.The sampling technique: at the beginning of the study, the research team held pre-study visits and held meetings with leaders of each stage to obtain the number of registered members and make appointments for interviews. The boda-boda stage leaders planned drivers´ schedules for interviews with the research team on the appointed date. On completion of the consenting processes, the research team completed face-to-face questionnaire interviews with drivers in the nearby quiet of each stage. Those who were not part of the interviews or had completed interviews continued to serve clients normally so that operations at the stage went minimally interrupted. The total number of drivers selected from each stage varied depending on the stage´s population size and availability of eligible members at the time of interviews but no more than three drivers were selected from each stage, and this was continued until the total sample size was realized. This restricted number of drivers selected per stage was to ensure that undeviating representation of participants across the four divisions was made. As for the victims of boda-boda accidents, the research team collected data from the Surgical Units of Gulu Hospital. The nurse in charge helped the research team identify victims of boda-boda accidents at the accident and emergency department and surgical wards of the hospital. The team then verified the participants using the selection criteria and performed the consenting process. Thereafter, the team held a face-to-face questionnaire interview with participants, attendants, and in charge of the unit.Data collection: we collected data from drivers and victims of boda-boda accidents using a questionnaire  [, p=072) .Data collection procedures: we collected data from drivers using a face-to-face questionnaire interview which lasted between 45-60 minutes in the nearby quiet of their workplaces , the time of the accident, where the accident occurred, what they considered was the causes/circumstances of the accidents, the site of injuries and using abbreviated injury scale (AIS), the injury was graded as minor, moderate and severe [d severe  and enteData management: all data obtained were double-checked and entered in Excel software and later exported to STATA version 14.1 for cleaning and analysis [analysis . Data enanalysis  was usedData analysis: to establish factors associated with boda-boda accidents and injuries in Gulu Municipality, we conducted univariate, bivariate and multivariable logistic regression analyses with boda-boda accidents as the outcome variable [variable . All varvariable ,3,5-7,21The principles of the model building were followed until we obtained the final statistical model for this study . We usedWhere: t = number of confounder adjustment variableThe strata were made in those that caused accidents and those that did not in the four divisions of Gulu Municipality .For each accident variables.For the pairs of responses.Using the statistical model-building methods mentioned above, four statistical models were developed. The first model examined the relationship between boda-boda accidents and income levels of drivers in the four divisions of Gulu Municipality. The second model was the associated factors with accidents for example age and work experience. The third model had levels of education, tribe, religion, marital status, and the number of children of boda-boda drivers. Note that in models two and three, none of the variables featured in any relationship with boda-boda accidents and were considered confounding variables in the model. The fourth model introduced the main occupation of drivers, availability of driving permits, ownership of the motorcycle, and knowledge of road safety rules.Determination on how poor monthly incomes among boda-boda drivers were associated with accidents among drivers in Gulu Municipality: first, we explored the relationship between boda-boda accidents and monthly incomes using a locally weighted scatterplot smoothing (lowess) (1) and exp(β2) then giving the odds ratios for the poorest and richest terciles compared to the middle tercile respectively. We added covariates (x) to check the sensitivity of our model. The first covariates we added were Gulu Municipal divisions, age, and work experience. Secondly, we added covariates from socio-demographic characteristics  and later, tribe, religion, and level of education. Finally, we added covariates about work conditions namely, driving as the main occupation the availability of valid drivers´ permits, and ownership of the boda-boda motorcycle.(lowess) . The lowThe final analysis showed that the logistic regression model remained stable, and analysis observed that irrespective of adding and removing confounding variables from the model, the model showed that poor monthly incomes were statistically and significantly associated with boda-boda accidents in Gulu Municipality  and particularly those from Pece division . However, drivers with better incomes more than UGX400,000/= were less likely involved in boda-boda accidents .Sensitivity analysis and model validation: we conducted sensitivity analysis and model validation of our findings on the understanding that: 1) all models possess inherent limitations for generalization; 2) there were assumptions made in making causal inferences with the available data; 3) it helped in checking the model fit and performances, especially controlling for confounding variables, for example, the reported information on accidents by boda-boda drivers [2=6.4035, p=0.094); an indication that the observed distribution of participants in the two datasets was likely due to chance; a reflection of the goodness of fit between reported and observed variables [ drivers . This wa drivers ,33. In aariables .2) =6.4035; p=0.092.To ascertain whether there were any misclassifications of data due to social desirability biases , a seconEthical approval: this study was approved by a local IRB ) and the Uganda National Council of Science and Technology (UNCST). Written informed consent was obtained from each selected participant before administering the questionnaire which contained details on the purpose, objectives, and benefits of the research to road users and policymakers. In addition, the research team assured participants that confidentiality of their information and the security of their data would be maintained. Only the principal investigator had access to their personal information that was de-identified, anonymized, and remained confidential.Overall, the questionnaire response rate for this study was 200/201(99.5%).Socio-demographic characteristics of boda-boda drivers and victims of accidents: this study showed that socio-demographic characteristics of boda-boda drivers in Gulu Municipality were young adults, consistent with other studies conducted in Uganda [n Uganda ,3,5-7,21n Uganda . In thisThe multivariable logistic regression analysis on factors associated with accidents among boda-boda drivers in Gulu Municipality: in 2) =6.4035; p=0.094).In The most significant findings from this study were that boda-boda drivers with poor monthly incomes were more prone to accidents, most victims of accidents were pedestrians and passengers from villages outside Gulu Municipality, drivers were young , and mosInterestingly, some boda-boda motorcycles we observed were in poor mechanical conditions with poor headlamp lights which could have contributed to the higher prevalence of boda-boda accidents and injuries in the evenings when it was dark. It was because of this that authors suggested that stricter enforcement and adherence to traffic rules could improve the situation in Gulu Municipality. Also, the authors argued that regular training of boda-boda drivers in Gulu by Ugandan police and Uganda Red Cross Society (URCS) on road safety rules and regulations were commendable and may have contributed to the adequate knowledge on road safety rules (85.5% versus 14.5%) and appropriate attitudes towards road safety rules and regulations (85% versus 15%) . These tIncreased costs of living and economic pressures among residents of Gulu Municipality: a recent survey conducted at the Gulu main market showed that food prices were considerably higher compared to most towns in Uganda [n Uganda ,38. Liken Uganda . Kampalan Uganda -41. ThesRapidly increasing population in Gulu Municipality: authors hint that higher food prices in Gulu could be attributable to the rapidly increasing population in Gulu Municipality without a matched food production. Increased number of students at Gulu University (a public university) and other higher institutions of learning for example Unyama National Teachers´ College (NTC), private Universities and academic institutions, for example, Uganda Management Institute (UMI), Gulu Clinical Officers´ Training School (Gulu COTS), University of the Sacred Heart, Cavendish University, and others have played a part [d a part -45. In ad a part -43. Thisd a part ,42 and md a part -41. AuthFew but expensive accommodations in Gulu Municipality: in further explanation to the economic pressures experienced in Gulu, charges for house rents in Gulu Municipality were higher compared to most towns in Uganda [n Uganda ,38. A mon Uganda . The numThe rapid urbanization of Gulu Municipality: in the current study, the multivariable logistic regression models showed that most boda-boda accidents occurred with drivers from the Pece division and most victims were pedestrians and passengers from villages outside Gulu Municipality . They have limitations based on the cross-sectional nature of the study design. A case-control or a cohort study would have produced much more powered results for the observations and outcomes we have reported. However, considering that we conducted the study in the four divisions of Gulu Municipality, the results among the four divisions and stages of the boda-boda drivers were comparable and provided generalizable information which was useful. Also, this current study was conducted on two occasions, making it closer to a longitudinal study, however, we got less than 30% of the boda-boda drivers in their respective states but the results of the first and second studies were nearly the same. The authors found that boda-boda drivers frequently change their stages for many reasons which we were unable to determine. Secondly, the study had a problem selecting drivers who were present and willing to take interviews. This may have caused a selection bias of our study participants. Only boda-boda drivers who were interested and did not have pressing issues were available for interviews, otherwise, boda-boda drivers preferred to do what was more gainful. This was however mitigated by working closely with the stage leaders by ensuring that appointments and confirmation for interviews were made in time and followed prudently. Thirdly, because boda-boda drivers were usually busy serving their clients, on bad days when clients were few, they preferred to return to their duties as quickly as possible even when interviews were already in progress. There were no monetary incentives to participants in this current study as funds for compensating the time spent at interviews were not available. This was however, mitigated by making appointments and receiving confirmations from the stage leaders in advance and ensuring that interviews were conducted quickly and yet exhaustively with the help of the stage leaders.Fourth, daily and monthly incomes recorded by boda-boda drivers were reported as average incomes and were not verified nor confirmed by any bank statements as most boda-boda drivers operated daily cash transactions. These reported daily/monthly incomes may not necessarily reflect the accurate financial status of each boda-boda driver but were used in calculations to determine its effects on the dependent variable (boda-boda accidents). We mitigated the uncertainties on incomes by getting corroborative information from leaders of stages who knew what each registered member earned daily, and we found that the information presented by the boda-boda drivers was consistent, comparable, and reliable. Fifth, recall bias and social desirability bias from this interviewer-administered questionnaire were also potential limitations for this study. For example, boda-boda drivers may have shillyshally to inform interviewers that they were being affected by other life unsettling issues such as unmet bank loans, undesirable family issues, and personal problems that may have affected their decision in driving boda-boda. These social problems were not explored in-depth but could form a basis for further studies on boda-boda drivers in Gulu Municipality. In addition, there could have been some more confounders that were residual that could have arisen due to misclassification of measured or reported variables on boda-boda drivers and victims of boda-boda accidents. These could include factors for example fatigue at work, a new environment in the newly constructed city, mechanical problems with the motorcycles, poor housing, personal loan obligations, pressures from families, and other personal liabilities. At this stage, we were unable to determine all these challenges. We hope that in the future we could conduct a more comprehensive study on the boda-boda drivers in Gulu Municipality and other cities in Uganda using a validated tool such as the Motorcycle Rider Behaviour Questionnaire (MRBQ) .Generalizability of information generated: results from this current study can be generalized to most major urban centers in Uganda especially the border towns in proximity to countries with weak economies for example South Sudan and the Democratic Republic of Congo (DRC). These border towns tend to have many boda-boda drivers, and many road traffic accidents are expected.Funding: this study was partly funded by the University of Oxford under the COOL project in addition to the support from individual authors. Funders were not part of the designs, data collection, and analysis of this data.The independent factors associated with road traffic accidents among boda-boda drivers in Gulu Municipality were poor monthly incomes and most accidents occurred in Pece division involving passengers and pedestrians from villages outside Gulu Municipality. Socio-demographic characteristics of drivers for example age, occupation, marital status, motorcycle ownership did not statistically affect the outcome of this study. These findings suggest that obehavioraloural issues need to be investigated among drivers and perhaps one of them could be stress resulting from the tough economic environment experienced by drivers due to the rapid urbanization of Gulu City. Several studies have viewed knowledge on road safety use, alcohol use, lack of driving permit, lack of helmets and other road safety wears, poor attitudes, and fatigue as the major drivers of boda-boda accidents in most settings. The paucity of information on the effects of economic pressures experienced by drivers  under enormous economic pressure in a rapidly growing urban environment needs further exploration. These authors suggest the following as recommendations: a more detailed and wide-ranging investigation into the economic conditions of boda-boda drivers in Gulu Municipality be undertaken with a view of conducting in-depth analysis on drivers and accidents. Secondly, there is nea edon to retrain boda-boda drivers on other income-generating activities as some divisions do not generate the required resources to meet their daily needs. Thirdly, the police and other law enforcement agencies should provide continuous education to the public  on proper road use considering that many accident victims were passengers and pedestrians from villages outside Gulu Municipality. Law enforcement agencies should remain vigilant and apprehend drivers that get involved in reckless driving. Finally, Motorcycle Riders Behaviour Questionnaire (MRBQ) should be used to conduct comprehensive studies on boda-boda drivers in Gulu Municipality and other municipalities in Uganda to determine behaviours of boda-boda drivers about accidents.Boda-boda accidents were the commonest reason for trauma admissions in many hospitals in Uganda;The prevalence of boda-boda injuries have been progressively increasing in Uganda;Boda-boda injuries were a major cause of early deaths and disabilities in Uganda.Poor monthly incomes and economic hardships among boda-boda drivers were associated with accidents and injuries in Gulu Municipality, Northern Uganda;Boda-boda drivers with better monthly incomes had lower risks of accidents and injuries in Gulu Municipality, Northern Uganda;Pedestrians and passengers from villages outside Gulu Municipality were more prone to boda-boda accidents and injuries.


Citrus maxima (Burm.) Merrill cv. ‘Hongmian miyou’ grafted onto Poncirus trifoliata) were inarched with ‘Pujiang Xiangcheng’ (a novel citrus rootstock cultivar recently selected from wild Citrus junos populations), and comprehensive analysis was performed to compare the inarched grafts and controls. Compared with incompatible grafts, the results revealed that inarching could recover the leaf metabolism balance, including reducing starch content, increasing chlorophyll content and restoring the cell structure. Additionally, our results corroborated that hormonal balance and hormone-related genes played a central role in inarching compatibility. Furthermore, the roles of Hsf4, ERF1, NCED3 and PYL were highlighted, and a model for explaining inarched grafts recovery invigoration was proposed. This study shed light on the mechanism of inarching regulation tree vigor and offered deep insights into the scion—rootstock interaction in citrus.Grafting is a technique that is widely used in citrus production. Graft incompatibility often occurs in the orchard. Inarching can effectively improve the vigor of incompatible grafts, but its mechanism remains poorly understood. Our previous studies investigated the scion—rootstock interaction of citrus and highlighted the role of hormonal balance and genes in abscisic acid biosynthesis regulation. To further elucidate the mechanism of inarched grafts rejuvenation, Hm/Pt combination ( Citrus is one of the most important fruit crops in the world, in terms of production. Grafting is widely used in citrus production, while some widely utilized rootstocks may still exhibit graft incompatibility in orchards ,2,3,4. LCitrus maxima (Burm.) Merrill) was grafted onto trifoliate orange (Poncirus trifoliata) (Hm/Pt combination) [It was found that graft incompatibility occurred when ‘Hongmian Miyou’ (ination) ,10,11,12ination) ,14. Thosination) ,16. Moreination) ,18,19,20ination) , its appCitrus junos populations [We previously found that incompatible grafts inarched with ‘Pujiang Xiangcheng’, a novel citrus rootstock cultivar recently selected from wild ulations , was ablC. maxima (Burm.) Merrill cv. Hongmian miyou (Hm) grafted onto P. trifoliata (Pt) as an incompatible graft combination (Hm/Pt), and Hm grafted onto C. junos Sieb ex Tanaka cv. Pujiang Xiangcheng (Pj) as a compatible graft combination (Hm/Pj) (We used  (Hm/Pj) a,b [9,10 (Hm/Pj) c. Etiola (Hm/Pj) a. The re (Hm/Pj) . The chl (Hm/Pj) d. We ins (Hm/Pj) e. Intrig (Hm/Pj) e.RNA-Seq was carried out, as described in the methods section, to determine the transcript levels of leaves from Hm/Pt, Hm/Pj and inarched plants (including SR and DR). STEM (Short Time-series Expression Miner) implemented unique methods to cluster, compare, and visualize gene expression data to study dynamical biological processes . We usedCitrus.Phytohormones regulate every aspect of plant development and response to biotic and abiotic stresses . The resTo better understand the molecular basis of the metabolic differences detected in the different graft combinations, the DEGs between Hm/Pt, Hm/Pj and inarched plants (including SR and DR) were identified. The results showed that, compared with the Hm/Pt, the number of DEGs of DR were 1660 upregulated and 1692 downregulated, and the number of DEGs of SR were 1763 upregulated and 2077 downregulated, respectively a,b. In oJAZ (Cg7g022240), AUX1 (Cg3g016080), AUX/IAA (Cg4g006940), GH3 (Cg8g003480) and PP2C (Cg8g023080). The results showed that the interaction between scion and rootstock could change the gene expression of the whole plant.Furthermore, there were 64 DEGs between DR with Hm/Pt and Hm/Pj, and 182 DEGs between SR with Hm/Pt and Hm/Pj, respectively . This map-value < 0.05)  and two DEGs were downregulated  (TIFY 10A) may have played an important role in hormone signal pathways. It was also the key gene in our previous study [Cg5g042950, Cg1g009760) were enriched in ‘Starch and sucrose metabolism’, which may have been responsible for starch accumulation in SR  a. Among  < 0.05) b. Out ofg015550) b. In thion in SR .r = 0.92) and starch (r = 0.88), and negatively correlated with chlorophyll (r = −0.88) and carotenoids (r = −0.85). This result suggested that the 4431 genes in the module ‘turquoise’ played important roles in the correction of rootstock-induced chlorosis in citrus . There were 19 DEGs  (MYB 15 (Cg2g015060) was only expressed in SR and DR, not in Hm/Pt and Hm/Pj  , which cnd Hm/Pj .NCEDs , PYL (Cg6g009750), PP2C (Cg8g023080) and TIFY-10A  . The results showed that ABA-related genes (NCED3 and PYL4) were significantly decreased in inarched plants (SR and DR) and Hm/Pj, compared with Hm/Pt. Meanwhile, the expression of PP2C showed no difference between inarched plants (SR and DR) and Hm/Pt, but significantly decreased levels in Hm/Pj   columnar gene candidate MdCoL and the AP2/ERF factor MdDREB2 were able to regulate ABA biosynthesis by activating the expression of MdNCED6/9 [Hsf (Heat stress factor) was able to connect ABA signaling and ABA-mediated stress responses [ERFs also worked synergistically with the change in auxin accumulation [NCED3 and PYL in graft compatibility [TIFY 10A showed high expression in inarched plants, which has been corroborated in previous studies [MYB 15 was only expressed in inarched plants, which could enhance sensitivity to ABA in Arabidopsis thaliana, leading to improved drought tolerance [Vitis vinifera) [Grafting has become highly important for citrus production. However, the mechanism of graft compatibility/incompatibility requires further investigation . Our pre grafts) . The appdNCED6/9 . The Hsfesponses ,26. In amulation . These gtibility   . TherefoMdWRKY9 mediated dwarfing by directly inhibiting the transcription of the DmDWF4 and reducing BR production in apple (M. domestica) [Citrullus lanatus) [Significant rootstock-induced change in hormone content has been reported in many species ,31,32. Omestica) . ABA cenlanatus) . In thislanatus) . The WGCHsf4 and ERF1. At the same time, several genes related to the auxin-transport and auxin-responsive pathways were differentially expressed. Additionally, the IAA level was increased and ABA level was decreased. Auxin signals, including AUX/IAA (Auxin/Indole-3-Acetic Acid), SAUR  and TIR (Transport Inhibitor Response), and ABA signals, including ABF, were regulated to coordinate plant growth and development [We refined the working model we previously proposed by using the evidence discussed above to gain insights into the citrus rootstock–scion interaction. Etiolater plant of Hm/Pt, inarched with ‘Pujiang Xiangcheng’ induced the different expression of TFs, including elopment ,39. ThisC. maxima (Burm.) Merrill cv. Hongmian miyou grafted onto P. trifoliata as an incompatible graft combination (Hm/Pt), and ‘Hongmian miyou’ grafted onto C. junos Sieb ex Tanaka cv. Pujiang Xiangcheng as compatible graft combinations (Hm/Pj) [All plant materials were planted in the orchard of Sichuan Agricultural University, Chengdu, China. The climate was a subtropical, humid monsoon climate with 1012.4 mm annual mean rainfall and an annual mean temperature of 15.9 °C. The soil type was purple soil with pH ranging from 6.8 to 7.4. We used  (Hm/Pj) ,12. The 663 − 2.69A645, Chlorophyll b (Chl b) = 22.9A645 − 4.68A663, total Chlorophyll (T-Chl) = Chl a + Chl b, Carotenoid (Car) = (1000A470 − 2.05 Chl a − 114.8 Chl b)/245. The contents of soluble sugar and starch were measured using a Plant Soluble Sugar Content Assay Kit  and a Starch Content Assay Kit  according to the manufacturer’s protocols.Carotenoid and chlorophyll extraction was performed according to the method of Wellburn and Lichtenthaler . Five fuv/v) was used to extract endogenous phytohormones. Crude extract was condensed by vacuum evaporation and hormones were re-extracted by ethyl acetate at pH 3.0 [Three pools of leaves collected from 10 inarched or grafted plants were used for endogenous phytohormone determination. Based on our previous results [t pH 3.0 . The levSamples were fixed in 2.5% glutaraldehyde in 0.03 M phosphate buffer for 24 h, then dehydrated in an ethanol series  for 90 min each. The method of sample preparation was developed and validated by He et al. . The basC. maxima [After seven months of inarching or grafting, leaves of Hm/Pt, Hm/Pj, SR and DR from inarched plants were collected and stored at −80 °C. Equal, small amounts of leaves from five inarched or grafted plants were pooled together as one replicate. Three independent replicates were carried out for RNA extraction, as described previously . In addi. maxima . The num. maxima . The ave. maxima  with |lo® Green qPCR SuperMix  on a CFX96 TounchTM Real-Time PCR detection system . The PCR program was as described previously [−ΔΔCt method was utilized to calculate gene expression and β-tubulin gene was used as an internal control.To verify the authenticity of the transcriptional data, 13 DEGs were selected for qPCR validation. The primer sequences were designed using Primer Premier 5 software  and listed in eviously . The 2−ΔThe physiological and biochemical indexes were entered into Microsoft Excel 2020 for collation and calculation. Significant differences between grafted combinations were analyzed by Tukey’s method. Column bar and box and whiskers plots were generated using GraphPad Prism (v. 7.04).In conclusion, our results supported the hypothesis that incompatible grafts’ vigor could be rapidly increased by inarching with ‘Pujiang Xiangcheng’ rootstock. The inarched plants recovered metabolism balance, including decreased starch content in the leaf, increased chlorophyll content, and enhanced recovery in the cell structure when compared with etiolater trees. This observation led us to demonstrate the important role of hormone signal in inarch graft compatibility. Furthermore, we identified several regulator genes that played vital roles during the recovery process. The detail regulation network warrants further research. Finally, we proposed a working model of inarched grafts’ rejuvenation based on the evidence observed. This study illustrated the mechanism of scion–rootstock interaction from a diverse yet comprehensive set of perspectives.