SFFD), transcribed from mitochondrion and mediated by HDAC1, was identified using RNA sequencing. Mechanistically, HDAC1 knockdown significantly reduced the H3K27ac level at the −953/−661 region of SFFD to epigenetically inhibit its transcription. SFFD knockdown released miR-202-3p to reduce the expression of cyclooxygenase 1 (COX1), an essential rate-limited enzyme involved in prostaglandin synthesis. This reduction inhibited the proliferation and secretion of 17β-estradiol (E2) while promoting the apoptosis of GCs. Consequently, follicular development was arrested and sexual maturity was delayed. Taken together, HDAC1 knockdown-mediated SFFD downregulation promoted the apoptosis of GCs through the miR-202-3p-COX1 axis and lead to delayed sexual maturity. Our findings reveal a novel regulatory network modulated by HDAC1, and HDAC1-mediated SFFD may be a promising new therapeutic target to treat delayed sexual maturity.Abnormal sexual maturity exhibits significant detrimental effects on adult health outcomes, and previous studies have indicated that targeting histone acetylation might serve as a potential therapeutic approach to regulate sexual maturity. However, the mechanisms that account for it remain to be further elucidated. Using the mouse model, we showed that Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, downregulated the protein level of Hdac1 in ovaries to promote the apoptosis of granulosa cells (GCs), and thus arrested follicular development and delayed sexual maturity. Using porcine GCs as a cell model, a novel sexual maturity-associated lncRNA, which was named as the stimulatory factor of follicular development ( Sexual maturity is a complex transitional phase in which adolescents acquire sexual maturation and reproductive capacity , and itsKiss1, a sexual maturity-activating gene, through elevating the level of histone H3 acetylation. Notably, the acetylation level in the Kiss1 promoter is remarkably higher in proestrus animals compared to diestrus animals [IGF1 promoter in fetal ovarian granulosa cells (GCs) notably inhibit the synthesis of E2 and follicular development [HDAC3) in GCs elevates the level of H3K14ac and promotes the binding of SP1 in the Areg promoter to initiate transcription and follicular maturation [AMH promoter, leading to elevated histone H3K27ac and markedly increased the expression of AMH, a natural gatekeeper of follicle growth and female reproduction [Histone acetylation, the most extensively studied and appreciated modification, exhibits an essential role in modulating chromatin structure to regulate gene transcription . Previou animals . Decreaselopment . Downregturation . In GCs,oduction . These oThe growth and function of GCs exhibit a dominant role in follicular maturation and sexual maturity ,20. The SFFD). Next, we aimed to explore the molecular mechanisms which histone acetylation regulated SFFD transcription and how SFFD regulated sexual maturation by affecting the functions of GCs. The regulatory mechanism and effects of the SFFD-miR-202-3p-COX1 axis on the proliferation, apoptosis, and estrogen secretion of GCs, follicular development, and sexual maturation were verified. Our results provide a new piece of evidence for HDAC1-mediated SFFD in the treatment of delayed sexual maturity.To characterize the mechanisms for histone acetylation controlling sexual maturity, Trichostatin A (TSA), an HDAC inhibitor, was treated with mice to explore the effects of histone acetylation on follicular development and sexual maturity. The physiological mechanisms of porcine follicular development are more similar to those of humans than mice . Therefon = 12), DMSO (n = 12), and TSA (n = 12) group. Mice were treated with TSA  and an equal volume of DMSO for three weeks. Mice were injected 3 times weekly. Among them, 8 mice in each group were randomly selected for counting the age of sexual maturity  from the Guangdong medical laboratory animal center were randomly assigned to three groups: blank ( opening ), and th opening . The num2. GCs were transfected with vectors using LipofectamineTM 3000 Reagent . In total, 100 ng/mL GDF9  and 50 ng/mL BMP15  were used to treat GCs for 24 h.Fresh pig ovaries were collected from a slaughterhouse and maintained in phosphate-buffered saline (PBS) containing penicillin and streptomycin during transporting. Porcine GCs were extracted from the follicular fluid of 3–5 mm follicles with a syringe and washed with PBS. Then, the GCs were cultured in DMEM containing 10% fetal bovine serum and finally incubated at 37 °C under 5% CO−ΔΔct method, and the primer sequences used in pig and mouse are listed in The purified total RNA was used as a template for cDNA synthesis with the RevertAid First Strand cDNA Synthesis Kit . qPCR was conducted using the SYBR Green qRT-PCR Master with the Bio-Rad CFX96 Touch Real-Time PCR system. The procedure steps were as follows: 95 °C for 10 min, 40 cycles at 95 °C for 15 s, annealing at 60 °C for 30 s. The levels of genes were calculated with the 2The process and details of RNA-seq were reported in our previous study . DiffereTM Edu Kit . Briefly, GCs were treated with 50 µM EdU at room temperature for 2 h, 80% acetone for 30 min, 0.5% Triton X-100 for 10 min, 1 × Apollo for 30 min, and Hoechst for 30 min.An EdU assay was conducted to detect the proliferation of GCs using the Cell-LightFlow cytometry was conducted to detect the apoptosis of GCs using the Annexin V-FITC Apoptosis Detection Kit . Briefly, GCs were incubated with Annexin V-FITC and propidium iodide for 15 min. The Caspase 3/7 Activity Assay was also performed to detect the apoptosis of GCs using the Caspase 3/7 Activity Assay Kit . A total of 50 μL of GCs lysis in 96-well plates was added along with 45 μL 2xReaction buffer and 5 μL of Ac-DEVD-pNA for 2 h. An absorbance of 405 nm was detected.The mouse E2 , progesterone (P4) (ml001945), and androgen (ml037278) ELISA kit were used to measure serum E2, P4, and androgen levels, respectively. Porcine E2 (ml002366) was used to measure the level of E2 secreted by porcine GCs. Briefly, 100 μL of HRP was added to the wells with 50 μL of standard and samples and incubated for 60 min at 37 ° C and 50 μL substrate A and B for 15 min. An OD value of 450 nm was obtained.n = 20) were randomly assigned into four groups: LV-NC, LV-lnc SFFD, sh-NC, and sh-lnc SFFD. Then, 1 × 107 TU of synthesized lentiviral vectors (LV-lnc SFFD and sh-lnc SFFD)  was delivered once a week for 3 weeks according to our previous work [Three-week-old female C57BL/6J mice . Briefly, the ovaries in paraffin sections were treated with xylene, ethanol, protease K, and TUNEL reaction mixture, respectively.SFFD promoter region. According to the PierceTM ChIP kit , the chromatin fragments were incubated with H3K27ac antibody  and IgG antibody , and the purified DNA was used for qPCR analyses; the primers are listed in ChIP was used to detect the H3K27ac level in the SFFD in GCs was detected using lncRNA FISH probe mix and matching kits . The GCs were treated with 4% paraformaldehyde, 0.5% Triton X-100, the SFFD probe, and DAPI.The localization of SFFD and miR-202-3p, the SFFD 3′ untranslated region (UTR) segment (lnc SFFD-WT) and the mutation sites of the seed sequence (lnc SFFD-MUT) were established and inserted into the pmirGLO-reporter  with Sac1 and Sall endonuclease. Then, lnc SFFD-WT/MUT and mimics NC/miR-202-3p mimic were co-transfected into GCs for 48 h, respectively. The luciferase activity was performed using a luciferase detection kit .To assess the binding between COX1 and miR-202-3p, the COX1 3′ UTR with binding sites of miR-202-3p, COX1 3′ UTR WT plasmid (COX1 3′ UTR-WT), and COX1 3′ UTR MUT plasmid (COX1 3′ UTR-MUT) were established, and COX1-WT/COX1-MUT plasmids and mimics NC/miR-202-3p mimic were co-transfected into GCs cultured in 24-well plates for 48 h, respectively.To assess the target between The total protein was extracted from GCs or ovaries using RIPA lysis buffer  and quantified using a BCA protein assay kit . A total of 20 µg of sample protein after being electrophoresed on 4–20% SDS-PAGE gels was transferred to polyvinylidene difluoride (PVDF) membranes. Then, the PVDF membranes were sealed with 5% skimmed milk for 1 h at room temperature and incubated with the primary antibody at 4 °C overnight: HDAC1 , HDAC2 , HDAC3 , PCNA , CCND1 , CASP3 , HSD17B1 , and Tubulin . Secondary antibodies were incubated for 2 h at room temperature. ImageJ was used to measure the bands.SFFD was performed using the 5′/3′-RACE Kit, 2nd Generation . To determine the coding ability of SFFD, the GFP-WT, GFP-MUT, and Open Reading Frame of SFFD with GFP-MUT were inserted into the pcDNA3.1 vector. The primers and oligonucleotide sequences are listed in The full-length amplification of t-test was used to measure differences. p < 0.05 or p < 0.01 were considered statistically significant.Data are exhibited as mean ± standard deviation (SD). Student’s p < 0.01, n = 8) (p < 0.05) and androgen (p < 0.01) but depressed the concentration level of E2 (p < 0.05), suggesting that the follicular development was blocked (p < 0.01) but displayed an insignificant effect on the protein levels of Hdac2 and Hdac3.To explore the role of histone acetylation on follicular development and sexual maturity, an HDAC inhibitor, TSA, was delivered into mice. Compared with DMSO  A,B. We f blocked C. Moreov blocked D. Especi blocked E. The TU blocked F. Immuno blocked G and Wes blocked H showed HDAC1 in GCs, the porcine GCs were treated with TSA and HDAC1-siRNA. The results showed that the viability , CDK1 (p < 0.01), CCNA1 (p < 0.05), and CCND1 (p < 0.01) related to cell proliferation (CASP3 (p < 0.01), CASP8 (p < 0.05), and BAX (p < 0.01) (HDAC1 downregulation markedly elevated the protein level of CASP3 (p < 0.05)  were constructed for HDAC1 knockdown. Notably, HDAC1-siRNA2 markedly decreased the mRNA (p < 0.01) and protein (p < 0.05) levels of HDAC1 (HDAC1 knockdown. We found that HDAC1-siRNA notably decreased the mRNA levels of PCNA (p < 0.05), CDK1 (p < 0.05), and CCND1 (p < 0.01) (CASP3 (p < 0.01) and CASP8 (p < 0.05) (HDAC1-siRNA markedly elevated the protein level of CASP3 (p < 0.05)  E. Moreov < 0.05) F. Subseq < 0.05) G showed ytometry H and Casytometry I furtherof HDAC1 J and was < 0.01) K and pro < 0.05) L. Moreov < 0.05) M. EdU st < 0.05) N showed ytometry O and theytometry P furtherHDAC1 downregulation prompted us to investigate the underlying mechanisms. Specially, the transcriptomes of GCs treated with HDAC1-siRNA (named H) and siRNA-NC (named NH) were analyzed, and 43 differential expression lncRNAs were obtained  and located at chr MT (mitochondrion): 1548–2026. Subsequently, we found the pcDNA3.1-ORF with GFP-MUT did not show GFP fluorescence , region 2 (−1288 to −1031 bp), region 3 (−953 to −661 bp), region 4 (−639 to −282 bp), and region 5 (−279 to −37 bp), was detected in GCs treated with HDAC1-siRNA , CDK1 (p < 0.01), CCNA1 (p < 0.05), CCND1 (p < 0.01) (CYP11A1 (p < 0.01), HSD17B1 (p < 0.01), and HSD3B1 (p < 0.01) (CASP3 (p < 0.01), CASP8 (p < 0.05), and CASP9 (p < 0.05) (SFFD overexpression remarkably decreased the protein level of CASP3 (p < 0.05) and did not show significant effects on the protein levels of CCND1 and HSD17B1 (SFFD knockdown markedly decreased the mRNA levels of PCNA (p < 0.01), CDK1 (p < 0.05), CCND1 (p < 0.05) (CYP11A1 (p < 0.05), HSD17B1 (p < 0.01), and HSD3B1 (p < 0.01) (CASP3 (p < 0.01), CASP8 (p < 0.01), and CASP9 (p < 0.05) (p < 0.05)  A, CYP11A < 0.01) B but dec < 0.05) C, and mi HSD17B1 D. SFFD k < 0.05) E, CYP11A < 0.01) F but inc < 0.05) G and the < 0.05) H, and thg effect I. Convery effect J. The EL effects K. Flow cpoptosis L and Caspoptosis M of GCs,poptosis N and Caspoptosis O of GCs,COX1 participates in the miR-202-3p-mediated pathway in GCs, three small interfering RNA  were used for COX1 knockdown. Among them, COX1-siRNA1 exhibited the most obvious inhibitory effect on the expression of COX1 (PCNA (p < 0.01), CCND1 (p < 0.05), and HSD17B1 (p < 0.05) (CASP3 (p < 0.05), CASP8 (p < 0.05), and CASP9 (p < 0.01) (PCNA (p < 0.01), CDK1 (p < 0.05), CCNA1 (p < 0.05), CCND1 (p < 0.01), CYP11A1 (p < 0.01), HSD17B1 (p < 0.01), and HSD3B1 (p < 0.01) (CASP3 (p < 0.01), CASP8 (p < 0.05), and CASP9 (p < 0.05)  B,C and e < 0.01) D. But th < 0.01) B,C, whil < 0.05) D, and COstaining E and ELIstaining F furtherytometry G exhibitSFFD was found to exist in mouse ovaries detected by FISH  and Hsd17b1 (p < 0.05) and inhibited the mRNA level of Casp3 (p < 0.05) (p < 0.05) (SFFD markedly reduced the mRNA levels of Pcna (p < 0.01), Cdk1 (p < 0.05), and Ccnd1 (p < 0.05) and promoted the mRNA levels of Casp3 (p < 0.01), Casp8 (p < 0.05), and Casp9 (p < 0.05) (SFFD significantly decreased the protein level of Ccnd1 (p < 0.05) and increased the protein level of Casp3 (p < 0.05) , sh-lnc SFFD markedly delayed sexual maturity, while LV-lnc SFFD did not show a significant effect on sexual maturity compared with LV-NC   E; it als < 0.05) F. Conver < 0.05) E. Sh-lnc < 0.05) F. Moreov, n = 5) G. The E2ecreased H. Notabl reduced I. TUNEL s of GCs J.HDAC1-siRNA, respectively. Both TSA-mediated HDAC1 knockdown and HDAC1-siRNA prominently arrested the proliferation of GCs (HDAC1 reduces proliferation and enhances apoptosis in various cell types [HDAC1 is involved in sexual maturity by mediating the apoptosis of GCs.At present, the epigenetic regulation mechanism of sexual maturity mainly focuses on DNA methylation. Lomniczi et al. found that the inhibition of DNA methylation resulted in rats failing to ovulate and a delay in sexual maturity . Howevern of GCs G,N and en of GCs H,O. Consll types . It is cHDAC1 remarkably decreased the expression of SFFD (HDAC1 epigenetically regulates the H3K27ac level at the gene’s promoter [HDAC1 inhibited the expression of SFFD, we analyzed the histone acetylation of the SFFD promoter and found that HDAC1-siRNA significantly decreased the H3K27ac level in the −953/−661 region at the SFFD promoter (HDAC1 exhibits deacetylase activity to inhibit transcription [HDAC1 [HDAC1 knockdown inhibits the expressions of super-enhancers associated with oncogenes through decreasing the H3K27ac level [HDAC1 knockdown leads to reduced levels of H3K27ac in the SFFD promoter may be the presence of a super-enhancer in the SFFD promoter region. But the mechanism of this needs to be further explored. The level of H3K27ac is significantly induced during follicular maturation in mice [ROCKI reduces the expression of MARCKS through facilitating the HDAC1-mediated removal of H3K27ac [SFFD.LncRNAs are generally considered to be transcripts that have over 200 nucleotides with no protein coding ability . It has  of SFFD A,B. Epigpromoter ,43. To epromoter I. It hascription . H3K27acn [HDAC1  and partn [HDAC1 . Consistac level . Therefo in mice . LncRNA  H3K27ac . These oSFFD was found to promote the proliferation (CCND1 and HSD17B1, respectively, and regulates the expression of CASP3 to inhibit the apoptosis of GCs (SFFD, the localization of SFFD in GCs was explored. The results showed that SFFD existed in both the cytoplasm and nucleus, but it was more abundant in the cytoplasm (SFFD may exert its cellular functions by acting as a sponge for miRNA. Through bioinformatics tools, miR-202-3p was predicted to be one of the potential targets of SFFD, and the target relationship between them was further verified using RNA pull-down (CCND1 in human Sertoli cells [SFFD. MiRNAs, as a vital epigenetic regulator, are evolutionarily conserved and exert an inhibitory effect on the expression of mRNA via a base–pairing interaction with the 3′ UTRs of target mRNAs [COX1 and miR-202-3p was verified through dual luciferase analysis (COX1, one of the rate-limiting enzymes of prostaglandin synthesis, exhibits a vital role in cellular proliferation [COX1-siRNA partially attenuated the pro-proliferation and anti-apoptotic effects of the miR-202-3p inhibitor (HDAC1 and SFFD in porcine GCs (HDAC1-mediated SFFD is involved in follicular development and sexual maturation. To characterize the in vivo function of SFFD, SFFD overexpression and knockdown were simulated by the direct injection of lentiviral in mouse ovary. We found that SFFD knockdown notably blocked the follicular maturation (SFFD in ovarian follicles and sexual maturation.In the present study, feration I,J and Eferation K of GCs s of GCs L,N. The ytoplasm E,F. Therull-down J and duaull-down K. MiR-20li cells . An elevli cells . In mousli cells . Consistet mRNAs ,53. In tanalysis P. COX1, feration , apoptosferation , and folferation  and deveferation . In thisnhibitor E,G. GDF9cine GCs B,C, whicturation I, signifturation H, and maturation G. These HDAC1 knockdown markedly reduces the H3K27ac level of the −953/−661 region to epigenetically inhibit the transcription of SFFD. SFFD functions by targeting miR-202-3p to elevate the expression of COX1, enhances proliferation and E2 secretion, and inhibits the apoptosis of GCs to regulate follicular development and sexual maturity (SFFD mediated by HDAC1 may serve as a potential molecular therapeutic approach for sexual maturity.In conclusion, our findings demonstrate that maturity . Collect


Begomoviruses are mainly transmitted by whiteflies. However, a few begomoviruses can be transmitted mechanically. Mechanical transmissibility affects begomoviral distribution in the field.In this study, two mechanically transmissible begomoviruses, tomato leaf curl New Delhi virus-oriental melon isolate (ToLCNDV-OM) and tomato yellow leaf curl Thailand virus (TYLCTHV), and two nonmechanically transmissible begomoviruses, ToLCNDV-cucumber isolate (ToLCNDV-CB) and tomato leaf curl Taiwan virus (ToLCTV), were used to study the effects of virus-virus interactions on mechanical transmissibility.Nicotiana benthamiana and host plants were coinoculated through mechanical transmission with inoculants derived from plants that were mix-infected or inoculants derived from individually infected plants, and the inoculants were mixed immediately before inoculation. Our results showed that ToLCNDV-CB was mechanically transmitted with ToLCNDV-OM to N. benthamiana, cucumber, and oriental melon, whereas ToLCTV was mechanically transmitted with TYLCTHV to N. benthamiana and tomato. For crossing host range inoculation, ToLCNDV-CB was mechanically transmitted with TYLCTHV to N. benthamiana and its nonhost tomato, while ToLCTV with ToLCNDV-OM was transmitted to N. benthamiana and its nonhost oriental melon. For sequential inoculation, ToLCNDV-CB and ToLCTV were mechanically transmitted to N. benthamiana plants that were either preinfected with ToLCNDV-OM or TYLCTHV. The results of fluorescence resonance energy transfer analyses showed that the nuclear shuttle protein of ToLCNDV-CB (CBNSP) and the coat protein of ToLCTV (TWCP) localized alone to the nucleus. When coexpressed with movement proteins of ToLCNDV-OM or TYLCTHV, CBNSP and TWCP relocalized to both the nucleus and the cellular periphery and interacted with movement proteins.Our findings indicated that virus-virus interactions in mixed infection circumstances could complement the mechanical transmissibility of nonmechanically transmissible begomoviruses and alter their host range. These findings provide new insight into complex virus-virus interactions and will help us to understand the begomoviral distribution and to reevaluate disease management strategies in the field. Potyviridae family. The coinfection of viruses results in frequent maize death and negligible yields. Although important advances have been made toward understanding the biology of individual viruses, little attention has been given to the investigation of intrahost interactions during viral coinfections.To study virus-host interactions, viral infections are usually conducted with the use of a single infection in the laboratory. Viral infections in nature can occur as part of mixed infections involving more than one type of virus. The results of a field survey between 2008 and 2009 in Taiwan indicated that more than 41% of virus-infected tomato were coinfected by more than two different viruses . A numbeMultiple infections cause a variety of intrahost virus-virus interactions. These interactions can result in the development of novel phenomena and alter the genetic structure of viruses . A studyin planta virus-virus interactions based on various plant responses, including cross-protection, synergistic and antagonistic interactions, recombination, heteroencapsidation, and gene silencing  . In thisN. benthamiana, cucumber (Cucumis sativus cv. MY02363), oriental melon (Cucumis melo var. makuwa cv. Silver Light), and tomato (Solanum lycopersicum cv. ANT22). Cucumber and oriental melon seeds were purchased from Known-You Seed Company . Tomato seeds were kindly provided by Dr. Wen-Shi Tsai . The resulting seedlings were transplanted into pots one week after germination. Plants that had been transplanted for one to five weeks were subjected to virus inoculation . The inoculated plants were maintained in a greenhouse located at the National Chung Hsing University .The plants used in this study included g  for 10 min, after which the Agrobacterium cells were resuspended in 20 ml of infiltration media  and then incubated at room temperature for 2 h before infiltration. Agrobacteria carrying the infectious DNA-A or DNA-B constructs were coinjected in equal amounts into leaves of N. benthamiana.The preparations of the infectious clones of ToLCNDV-CB, ToLCNDV-OM, ToLCTV, and TYLCTHV were carried out as described in our previous studies . AgroinfN. benthamiana for the single virus-infecting inoculum. Infectious clones for two viruses  were mixed in equal amounts for agroinfiltration as mixed-infection inocula, namely, “virus A/virus B”.Viral inocula were prepared in single and mixed-infection types. Infectious clones for one virus were agroinfiltrated into N. benthamiana plants were collected as inocula for mechanical inoculation. The leaf samples were ground in 10 mM potassium phosphate buffer (pH 7.0) at a 1:20 ratio (weight/volume). The resultant sap was inoculated onto N. benthamiana, cucumber, oriental melon, and tomato plants by rubbing with carborundum powder (400-500 mesh). Afterward, the inoculated leaves were washed with distilled water to remove the inoculation buffer and carborundum powder from the leaf surface. The inoculated plants were subsequently grown in an insect-free greenhouse. Two to four weeks after inoculation, systemic leaf samples of the mechanically inoculated plants  were collected for virus detection.Symptomatic leaves of agroinfiltrated Mechanical inoculation involved three inoculation types. The first one was a single-virus inoculation type with inocula derived from the diseased plants infected by only one virus isolate; the plants were labeled “virus A” or “virus B”. The second one was a mixed-infection inoculation type with inocula derived from the diseased plants infected by two different viruses; the plants were labeled “virus A/virus B”. The third type involved two viruses coinoculated (coinoculation type) with an inoculum mixture derived from two individual diseased plants infected by different viruses; the plants inoculated in this manner were labeled “virus A+virus B”.via microprep buffer containing a mixture of three kinds of buffer, namely, DNA extraction buffer , nuclei lysis buffer , and 5% sarkosyl, at a 5:5:2 volumetric ratio. The collected leaf samples (50-100 mg per sample) were ground in 750 μl of microprep buffer. The ground sample solution was incubated at 65 °C for 30-120 min. Five hundred microliters of chloroform/isoamyl (24:1) mixture was then added to the sample solution, which was then vortexed for 0.5-1 min. The homogenized samples were then centrifuged at 12000 × g  for 5 min. The top layer of the centrifuged suspension was pipetted (500 μl) into a new 1.5 ml Eppendorf tube. Afterward, 500 μl of isopropanol was added to the tube, after which the contents were mixed gently. The samples were pelleted by centrifugation at 12,000 × g  for 5 min. The DNA pellet was subsequently washed with 70% ethanol and then dried in an oven for 1-2 min to remove ethanol. The DNA pellet was subsequently resuspended in 50 μl of sterilized distilled water and kept at -20 °C for further analysis.Plant total DNA was extracted following the methods of ProTaq Plus DNA polymerase  and reaction buffer. The conditions used for amplification were as follows: one cycle at 95°C for 5 min followed by 35 cycles of 95°C for 1 min, 50°C for 1 min and 72°C for 1 min. The PCR products were analyzed via electrophoresis involving a 0.8% agarose gel for 30 minutes with a voltage of 120 V.The detection process was carried out using specific primers  with different wavelength channels, namely, CFP (excitation 405 nm/emission 460-500 nm), YFP (excitation 488 nm/emission 530-630 nm), and FRET (excitation 405 nm/emission 530-630 nm), and analyzed using FV31S-SW software (Olympus).Full-length begomoviral genes  were amplified with specific primers  to genervia coinfection with ToLCNDV-OM. Symptomatic leaves of agroinfiltrated plants were collected as inocula for mechanical inoculation of N. benthamiana. Severe leaf curl symptoms were observed for N. benthamiana mechanically inoculated with ToLCNDV-OM, ToLCNDV-OM/ToLCNDV-CB, and ToLCNDV-OM+ToLCNDV-CB at 14 dpi, while no symptoms were observed in N. benthamiana inoculated with ToLCNDV-CB alone  and ToLCTV  were used for mechanical transmission analyses. Severe mosaic and leaf curl symptoms were observed in TV alone Table 2.ToLCNDV-OM and ToLCNDV-CB were isolated from cucurbit plants, while TYLCTHV and ToLCTV were isolated from tomato plants. The results of agroinfiltration of ToLCTV alone showed that oriental melon was not a host of ToLCTV  sequence was fused to the N-terminus of the MPs of ToLCNDV-OM (YFP-OMMP) and TYLCTHV (YFP-THMP). Yellow fluorescence (excitation 488 nm/emission 530-630 nm) emitted from the YFP-OMMP fusion protein was observed in the cells, and the fluorescence signal was evenly distributed along the periphery of the cells  analyses were conducted with fusion proteins expressed in he cells Figure 5The protein-protein interactions were also analyzed with BiFC. The N half of YFP sequence was fused to the C-terminus of the MPs of ToLCNDV-OM and TYLCTHV to yield OMMPnYFP and OMMPnYFP, respectively. The C half of YFP sequence was fused to the C-terminus of the NSP-coding gene of ToLCNDV-OM (OMNSPcYFP) and ToLCNDV-CB (CBNSPcYFP) and the CP gene of ToLCTV (TWCPcYFP). Interactions between OMMPnYFP and OMNSPcYFP were used as positive controls, and OMMPnYFP and cYFP empty vector (cYFP) were used as negative controls. The YFP fluorescence was observed in different combinations, OMMPnYFP/CBNSPcYFP, OMMPnYFP/TWCPcYFP, THMPnYFP/CBNSPcYFP, and THMPnYFP/TWCPcYFP, mainly along the periphery of the cells, but not in nucleus Figure 6Infections involving a mixture of viruses can potentially trigger complex virus-virus interactions. Except for recombination, virus-virus interactions modify the characteristics of the infecting viruses, such as tissue tropism, infection and movement facilitation and insect transmissibility . MechaniN. benthamiana and other crop species together with ToLCNDV-OM or TYLCTHV through coinoculation, as was ToLCTV. Based on our results, this complementation was observed in experiments involving coinoculation with both “virus A/virus B” and “virus A+virus B”. PCR detection and sequence analysis were used to confirm the presence of the infection viruses and rule out the possibility of contamination. These results corroborated that this complementation of mechanical transmissibility was not due to gene mutation or genomic recombination between the coinoculated viruses. In this study, the symptoms of the coinfected plants were not more severe than those of the single virus-infected plants , S. melongena, N. benthamiana, Datura stramonium, Lonicera japonica, Petunia hybrida, and Physalis floridana (loridana . In our loridana . The reploridana . An adaploridana . Early eloridana . Taking Based on a previous report by Tsai et al. , throughvia mechanical transmission is more likely than expected and consequently makes disease management more difficult. Complementation of mechanical transmissibility in sequential infection increased the rate of mixed infection in the field, and those complex interactions led to trait changes such as altered host range. Although our FRET and BiFC analyses revealed protein-protein interactions between different viral proteins, the virus-virus interaction leading to the alterations of mechanical transmission and host range may happen in other ways through an indirect or non-physical contact between begomoviruses. Further studies to elucidate virus-virus interactions will provide critical insights to aid in strategizing the control of viral spreading and reducing disease severity in the field.In this study, we tried to analyze the complementation of mechanical transmissibility between begomovirus infection mixtures. Our results indicated that complex virus-virus interactions occurred during mixed infection and that these interactions complemented the mechanical transmissibility and altered the host range of the begomoviruses. This complementation can occur between unrelated and different species of either monopartite or bipartite begomoviruses. These findings indicated that the spread of begomoviruses The original contributions presented in the study are included in the article/H-HC designed the methods, performed the experiments, interpreted the data, and wrote the original draft. DG designed and performed the experiments, interpreted the data, and revised the manuscript. C-JC interpreted the data and revised the manuscript. F-JJ administered the research project, planned and designed the research, interpreted the data, and revised the manuscript. H-HC and DG contributed equally to this work. All authors contributed to the article and approved the submitted version.


Mpox (earlier known as monkeypox) virus infection is a recognized public health emergency. There has been little research on the treatment options. This article reviews the specific drugs used to treat mpox virus infection and the vaccines used here. Instead of focusing on the mechanistic basis, this review narrates the practical, real-life experiences of individual patients of mpox virus disease being administered these medicines. We conducted a bibliometric analysis on the treatment of the mpox virus using data from several databases like PubMed, Scopus, and Embase. The research on this topic has grown tremendously recently but it is highly concentrated in a few countries. Cidofovir is the most studied drug. This is because it is indicated and also used off-label for several conditions. The drugs used for mpox virus infection include tecovirimat, cidofovir, brincidofovir, vaccinia immune globulin, and trifluridine. Tecovirimat is used most frequently. It is a promising option in progressive mpox disease in terms of both efficacy and safety. Brincidofovir has been associated with treatment discontinuation due to elevated hepatic enzymes. Cidofovir is also not the preferred drug, often used because of the unavailability of tecovirimat. Trifluridine is used topically as an add-on agent along with tecovirimat for ocular manifestations of mpox virus disease. No study reports individual patient data for vaccinia immune globulin. Though no vaccine is currently approved for mpox virus infection, ACAM 2000 and JYNNEOS are the vaccines being mainly considered. ACAM 2000 is capable of replicating and may cause severe adverse reactions. It is used when JYNNEOS is contraindicated. Several drugs and vaccines are under development and have been discussed alongside pragmatic aspects of mpox virus treatment and prevention. Further studies can provide more insight into the safety and efficacy of Tecovirimat in actively progressing mpox virus disease. While the world is finding ways to deal with SARS-CoV-2 and COVID-19, a novel threat of mpox has emerged in the human population. The World Health Organization (WHO) has declared this a health emergency, and the confirmed cases have risen to 84,916 and 81 deaths as of 20 January 2023. One hundred ten countries throughout the globe have reported cases . The mpoTreatment of the mpox virus is a new challenge for the entire healthcare system . There aThe bibliometric analysis involves using mathematical methods to study books and communication media . It is bIf we further look at the countries where research is ongoing in the field of mpox virus disease, we can notice a worrying trend . Though There is a clear trend in the funding patterns in research pertaining to the mpox virus . The NatUsing the results of the aforementioned search strategy, a bibliometric map of the relevant keywords was constructed using VOSviewer. We performed a co-occurrence analysis of keywords across several databases using whole counting. This helped us visualize the critical areas discussed in the sparsely available literature on the treatment of mpox. in-vitro activity against several DNA viruses like herpesvirus, poxvirus, polyomavirus, and papillomavirus  and those undergoing organ transplantation. It also demonstrates omavirus . It is uomavirus , acyclovomavirus , skin leomavirus , BK polyomavirus . We haveomavirus . We haveTecovirimat is the most commonly employed antiviral in patients with mpox infection. Initially identified in a high throughput screening, the United States FDA granted it approval in 2018, and it is indicated in smallpox disease . TecovirThe mpox virus enters the cytoplasm of human cells. After pre-processing, it replicates its DNA in Guarnieri inclusion bodies . Then, tin silico study has shown better efficacy against mpox virus with combination of atovaquone and TPOXX . The multinational randomized controlled trial has started recruiting. The second trial, which is based in Canada, is yet to start recruiting. Both are expected to be completed by the following year, i.e., 2023 .Cidofovir is used in diseases due to cytomegalovirus (CMV), herpesviruses, and several DNA viruses. It is indicated in certain CMV diseases in immunocompromised people  and has https://pubmed.ncbi.nlm.nih.gov/30397065/). Pharmacologically, it is an example of a hit-and-run drug. Administered intravenously, the serum concentration of the drug falls rapidly following the infusion, and it has a short plasma half-life of 2 h. However, the intracellular half-life of the active form is as high as 65 h. Cidofovir undergoes renal elimination, and this involves a critical drug-drug interaction. Probenecid blocks tubular secretion of this drug, thereby reducing its excretion and increasing its serum level  is a nucleotide analogue DNA polymerase inhibitor. It is a pro-drug composed of cidofovir conjugated to a lipid molecule. The lipid component resembles an endogenous lipid called lysophosphatidylcholine, allowing the molecule to enter the infected cells by taking on the natural lipid absorption mechanisms. Following absorption, the lipid molecule is broken down, releasing cidofovir for additional intracellular kinase phosphorylation to form cidofovir diphosphate, the active form of the drug. In contrast to cidofovir, brincidofovir does not act as a substrate for Organic Anion Transporter 1, which makes BCV less harmful to the kidneys. Therefore, brincidofovir has a higher safety profile for nephrotoxicity compared to cidofovir. Coming to preventive measures and adverse reactions of Brincidofovir, its administration requires continuous monitoring of hepatic function tests as it increases the serum transaminase and bilirubin levels. Other adverse effects seen are gastrointestinal side effects like diarrhea and vomiting. Pregnancy is ruled out before administering this drug as it is teratogenic in animal studies. Contraception is advised throughout the treatment and for 4 months after that. Brincidofovir is also known to have carcinogenic potential, so safety with handling is necessary. Brincidofovir is taken on an empty stomach or with a low-fat meal to increase the bioavailability of the drug. Drug-drug interactions are seen when used concomitantly with inhibitors of OATP1B1 and 1B3 (Organic Anion Transporting Polypeptide) like rifampin, erythromycin, and protease inhibitors like ritonavir as they increase its peak serum concentration, increasing the adverse events due to Brincidofovir .Results with Brincidofovir have not been promising. In the solitary study on human patients, all three patients had to discontinue treatment because hepatic impairment led to hospitalization prolonging. Other issues encountered were conjunctivitis, lower limb abscess, and neuropsychiatric symptoms . They weMany medical countermeasures are kept on hand in case of orthopoxviruses as mpox emerges. Although most instances of mpox are minor and self-limited, supportive treatment is often enough to treat them. Most patients have moderate sickness and recover without medical help, but in very unwell or immunocompromised people, antivirals or vaccinia immune globulin (VIG) may be utilized. According to the Centre for Disease Control and Prevention (CDC), supportive care is often sufficient for people with a mpox virus infection because no particular medicines are available. Mpox virus disease can be prevented and treated similarly to other orthopoxvirus infections, and unless proven differently, all confirmed orthopoxvirus cases should be managed as though they are mpox .In immunosuppressed patients exposed to mpox for whom ACAM2000 vaccination is contraindicated, VIG, an injectable preparation of hyperimmune globulin made from the pooled blood of smallpox vaccine recipients, may be considered. These people’s acquired antibodies against the smallpox vaccine are removed and purified. Additionally, VIG can treat vaccinia virus-related aberrant infections brought on by autoinoculation, eczema vaccinatum, or severe generalized or progressive vaccinia . VIG is Trifluridine is a fluorinated structural analogue of the DNA constituent thymidine. It acts by inhibiting DNA synthesis. It inhibits the enzymes involved in this process and may itself get incorporated into DNA. However, its action may lack selectivity. It has only been used as a topical preparation for the eye in cases of mpox virus infections. It is said to be safe when applied topically as eye drops. This is because it does not penetrate the intact cornea. However, in cases with corneal pathologies disrupting its structure, trifluridine may penetrate the cornea and be detectable in aqueous humor. Mild adverse events have been noted. These include transient local burning sensation, oedema of the eyelids, inflammation of the cornea, and allergy. It may be dosed at a 2-h interval till there is complete regeneration of epithelium in the cornea. Then, it may be administered once every 4 h for another 7 days. However, it is not prescribed for prolonged durations. In such cases where it has to be given beyond 3 weeks, alternative pharmacological options may be explored .Trifluridine has been used in mpox virus infection. However, its use in both the studies has been as an add-on agent. Overall, five patients received both Tecovirimat and local administration of Trifluridine. Though all these four patients varied greatly in their presentation, they were common in that all had ophthalmological manifestations of mpox virus disease too. Four of them recovered promptly and were discharged. One of them continues to develop worsening ocular symptoms and decreased visual acuity. No adverse events were reported with this drug .Researchers are exploring several new potential drug targets and therapeutic options for the treatment of mpox amidst concerns regarding both supply shortages and drug resistance . MutatioThe genetic material of all orthopoxviruses show similarity. The open reading frames responsible for viral protein synthesis are well-conserved in orthopoxviruses. This is especially manifested in the case of the VP37 protein. Tecovirimat targets this protein and has shown positive results in mpox. Therefore, novel drugs can be developed focusing on the VP37 protein . There ain-vitro and in-vivo studies involving orthopoxviruses including mpox virus -isonicotinoyl-N(2)-3-methyl-4-chlorobenzoylhydrazine. It has a similar mechanism of action, and inhibits envelopment, extracellular release and consequent propagation of disease in the human body. Though beneficial in l models . To add l models . This cain-vitro studies on orthopoxviruses . There are no approved vaccines specifically for mpox virus infection. The mpox virus and the variola virus  belong to the same genome of orthopoxvirus. Orthopoxviruses are known to share immunological cross-protection between them, but the evidence is not very strong. Immunity developed against smallpox may help protect against mpox virus infection too. Thus, smallpox vaccines are being repurposed for use in mpox virus infection .JYNNEOS (or Imvamune) is a third-generation vaccine. It is modified vaccine Ankara (MVA), manufactured in Denmark. It comprises a virus that is incapable of replicating. It is approved in the United States for both smallpox and mpox virus infection. 0.5 mL of the vaccine is delivered subcutaneously 4 weeks apart. Then, the person is said to have been vaccinated 2 weeks after the second dose. It is also approved by the World Health Organization and medical agencies in Europe and Canada for post-exposure prophylaxis. There are several trials that are undergoing to test this vaccine further. Intradermal administration has also been practiced . HoweverACAM 2000 is a second-generation vaccine. It is a derivative of the first-generation Dryvax vaccine . It diffThese vaccines have been associated with several adverse events including myocarditis and pericarditis . ImvamunSeveral other vaccines for mpox virus infection are under development or under study. Aventis Pastuer smallpox vaccine is being used under an investigational new drug protocol for smallpox. This may be later developed for use in mpox too . A JapanThis review elaborates on the different pharmacological treatment options for mpox virus infection. A bibliometric analysis was also carried out across several databases like PubMed, Scopus, and Embase. We observed that five drugs are mainly used for specific management of mpox disease in humans: tecovirimat, cidofovir, brincidofovir, trifluridine, and Vaccinia Immune Globulin. Tecovirimat has emerged as an exciting option with efficacy in progressive disease. No signals for safety concerns have been detected either. All other options are infrequently used. Cidofovir and its related compound brincidofovir are also used. The latter is linked with hepatic impairment, and treatment had to be discontinued in all three cases in a study. Vaccinia immune globulin has not been used much and is mainly preferred for other indications like post-vaccine complications. Trifluridine is successfully used as an add-on treatment option in patients with ocular manifestations of the mpox virus.According to the interim guidelines by CDC for treating mpox virus infection, treatment should not be considered across all cases. It should only be considered based on clinical features and individual baseline risk. Clinically, severe disease and involvement of areas of the body that can potentially cause serious complications are conditions for treatment consideration. Pharyngeal lesions may lead to dysphagia and lack of control of secretions. Rectal involvement may lead to severe proctitis and pain. Treatment should also be considered in high-risk individuals like immunocompromised, pregnant, lactating, children, and those with dermatological diseases that affect cutaneous integrity .These drugs still need to be adequately tested in well-designed studies on patients with monkeypox infection. The main reason behind the lack of such studies might be feasibility issues. Further studies, including randomized controlled trials like these , must confirm the results and optimize the dosage range.


Diabetes and hypertension are the 2 major causes of endstage renal disease. The rate of chronic kidney disease (CKD) secondary to diabetes and/or hypertension is on the rise, and the related health care costs represent a significant economic burden.To quantify from a health system perspective the incremental direct all-cause health care costs associated with a diagnosis of CKD in patients with diabetes and/or hypertension.An analysis was conducted of medical claims and laboratory data with dates of service between January 1, 2000, and February 28, 2006, from a managed care database for approximately 30 million members enrolled in 35 health plans. Each patient's observation period began on the date of the first diabetes or hypertension diagnosis (index date) and ended on the earlier of the health plan disenrollment date or February 28, 2006. Inclusion criteria were continuous insurance coverage in the 6 months prior to the index date and during the observation period, age at least 18 years, and at least 2 claims less than 90 days apart with a primary or secondary diagnosis for diabetes or hypertension. Exclusion criteria were cancer, lupus, or organ transplantation or chemotherapy at any time during the observation period. CKD was defined as at least 1 claim with a primary or secondary diagnosis for CKD and at least 2 glomerular filtration rate values of below 60 milliliters per minute per 1.73 square meters of body surface area (60 mL/min/1.73m2) at any time during the observation period. Bivariate and Tobit regression analyses were conducted to compare patients who developed CKD versus those who did not for annualized (per patient per month [PPPM] multiplied by 12) direct, all-cause, health care costs, defined as standardized net provider payments after subtraction of member cost-share. These costs consisted of outpatient services, inpatient services, and pharmacy claims. A subset analysis of the post- versus pre-CKD medical costs was also conducted for cohorts of patients with at least 60 days of observation before and after the development of CKD; that analysis measured both all-cause costs and costs for services directly related to CKD treatment .11,531 patients with diabetes, 74,759 patients with hypertension, and 4,779 patients with both conditions were identified, of whom 123 (1.1%), 1,137 (1.5%), and 712 (14.9%), respectively, developed CKD during the observation period. The CKD group was older than the no-CKD group in each cohort . CKD was associated with significantly higher total direct all-cause health care costs, with unadjusted annualized per patient mean [median] cost differences of $11,814 , $8,412 , and $10,625 , respectively . The largest driver of the all-cause mean cost difference associated with CKD for each cohort was hospitalization cost . Among patients developing CKD, all-cause mean [median] annualized costs increased significantly following CKD onset . In the post-CKD period, costs directly related to treatment of CKD accounted for 9%-19% of all-cause medical service costs 9.2% for patients with diabetes.


Pathogens carried by insects, such as bunyaviruses, are frequently transmitted into human populations and cause diseases. Knowing which spillover events represent a public health threat remains a challenge. Metagenomic next-generation sequencing (mNGS) can support infectious disease diagnostics by enabling the detection of any pathogen from clinical specimens. mNGS was performed on blood samples to identify potential viral coinfections in human immunodeficiency virus (HIV)-positive individuals from Kinshasa, the Democratic Republic of the Congo (DRC), participating in an HIV diversity cohort study. Time-resolved phylogenetics and molecular assay development assisted in viral characterization. The nearly complete genome of a novel orthobunyavirus related to Nyangole virus, a virus previously identified in neighboring Uganda, was assembled from a hepatitis B virus–positive patient. A quantitative polymerase chain reaction assay was designed and used to screen >2,500 plasma samples from Cameroon, the DRC, and Uganda, failing to identify any additional cases. The recent sequencing of a US Center for Disease Control Arbovirus Reference Collection revealed that this same virus, now named Bangui virus, was first isolated in 1970 from an individual in the Central African Republic. Time-scaled phylogenetic analyses of Bangui with the related Anopheles and Tanga serogroup complexes indicate that this virus emerged nearly 10,000 years ago. Pervasive and episodic models further suggest that this virus is under purifying selection and that only distant common ancestors were subject to positive selection events. This study represents only the second identification of a Bangui virus infection in over 50 years. The presumed rarity of Bangui virus infections in humans can be explained by its constraint to an avian host and insect vector, precluding efficient transmission into the human population. Our results demonstrate that molecular phylogenetic analyses can provide insights into the threat posed by novel or re-emergent viruses identified by mNGS. Metagenomic next-generation sequencing (mNGS) of patient specimens enables the detection of any pathogen otherwise undetectable with conventional molecular methods. While its full clinical impact has yet to be realized, there are clear examples demonstrating that mNGS can lead to an actionable diagnosis and treatment . AnotherNairovirus, Orthobunyavirus, and Phlebovirus are arboviruses that infect vertebrates and are vectored by hematophagous flies, mosquitos, culicoid midges, and ticks (Viruses transmitted by insects (arboviruses) are frequent causes of acute febrile illness, encephalitis, rashes, and hemorrhagic fever. Bunyavirus genera including nd ticks . The reand ticks . As natund ticks . Not surnd ticks .Orthobunyavirus genus (Peribunyaviridae) consists of >220 species classified into 18 serogroups based on serological relatedness, as determined by complement fixation, neutralization, and hemagglutination inhibition  viral diversity study in 2017–9 as previously described . The invm2000 HIV-1 RealTime assay in serology-positive specimens. Specimens that were negative for HIV by serology and/or viral load testing were further screened with a research-use-only HIV-1/HBV/HCV multiplex polymerase chain reaction (PCR) assay conducted with the Abbott m2000sp  and CFX96 Real Time  instruments in pools as previously described , and hepatitis C virus (HCV) has been outlined previously , with exescribed . HBV virm2000sp system , reverse transcribed using SuperScript IV , converted into Nextera XT libraries, and sequenced on an Illumina MiSeq instrument at Abbott Laboratories as previously described . Metagenomic libraries were prepared at the University of California San Francisco using the metagenomic sequencing with spiked primers (MSSPE) method as described previously . Brieflyescribed .de novo assembly. An additional in-house pipeline utilizing psiBLAST  and nucleocapsid (Cy5) genes of Bangui orthobunyavirus. Primers and probes were based on the CHRB594 sequence: RdRp Forward Primer: 5ʹ-ACCAGTAGCAAGTCCTGTATTA-3ʹ; RdRp Reverse Primer: 5ʹ-TTGCTATCTTACACTTCCTCTG-3ʹ; RdRp Probe oligo: 5ʹ-FAM-TGGCTGCTTTGAACTCTTGGAAATCACC-BHQ1-3ʹ; Nucleocapsid Forward Primer: 5ʹ-GCTTGGCCAACTCAGATTTAACA-3ʹ; Nucleocapsid Reverse Primer: 5ʹ-CTTTTCTTTCCACAGAGCAGCTT-3ʹ; and Nucleocapsid Probe oligo: 5ʹ-Cy5-CCGCATCTCCATGTTTGACTATGTCCC-BHQ2-3ʹ.A multiplex RT-qPCR assay was developed on the Abbott In-vitro transcripts targeting the L segment (RdRp)  and the S segment (nucleocapsid)  were purified through Clontech Chroma Spin Column-100, size-confirmed on a 1.2 per cent formaldehyde agarose gel, and quantified by Qubit . A complete description of RT-qPCR assay volumes and cycling parameters for the m2000rt can be found in the m2000sp before the addition of quantitative polymerase chain reaction (qPCR) master mix and cycling on the m2000rt. Amplification curves were analyzed using MultiAnalyzer software.Target regions for assay development were cloned into pBlueScript and expressed from the T7 promoter using a MEGAscript T7 Transcription Kit  according to the manufacturer’s instructions. https://www.ncbi.nlm.nih.gov/nucleotide/): the first (Dataset A) contained 703 orthobunyavirus representatives and the outgroup Khurdun virus and the second (Dataset B) contained 22 known viruses from the Anopheles A/B and Tanga serogroups with published genomes for all three segments. Whole-segment nucleotide alignments and protein alignments were performed using Clustal Omega  phylogenetic inference from the protein alignments was performed using IQ-TREE version 2.1.3 . The Modggplot2  and ggtr  were conducted per genome segment. A comparison of the statistics from both the PS and SS methods was tabulated to compare the models  in the sequences of interest, two methodologies were followed. First, a root-to-tip analysis was used: ML trees and collection dates from Dataset B were provided as input to the programs TempEst and TreeTime to calculate an underlying temporal signal using the heuristic residual mean-squared and least-squares models, respectively . Second,e models .−5 substitutions/site/year; M = 6.789 × 10−5 substitutions/site/year; and S = 5.564 × 10−5 substitutions/site/year . The BEAGLE v.4.0 pre-release library  and CUDATo analyze the impact of both pervasive and episodic selection on the evolutionary process of the serogroups included in Dataset B, codon-based alignment of the polyprotein coding sequence of each viral segment and the corresponding isochronous MCC trees were used as input for the CODEML program of the PAML v.4.9 software package .2 and M8 as described by dN/dS, also called ω) were calculated for each site of the alignments, assuming a single average ω across the entire tree. The Bayes Empirical Bayes calculation of posterior probabilities for site classes was used to estimate the probabilities of sites under positive selection. As described previously (2 and M8) with models used to detect neutral selection (M1 and M7) , an analysis contrasting models M0 and M3 was conducted  . Only ca and M7) . In addionducted .ω along all branches (0 < ω < 1 and ω = 1) and allows for an additional class only along prespecified foreground branches for codons under positive selection (ω > 1). This model was contrasted with the null Model A1, which does not allow ω > 1 along foreground branches . The significances of the LRTs were calculated assuming that twice the difference in the log ML between the two models was distributed as a f models .Orthobunyavirus genus, including California encephalitis virus, Bunyamwera virus, Tacaiuma virus, Salt Ash virus, and Wyeomyia virus. This specimen was drawn on June 2017 from a 40-year-old male from Kinshasa. He was HBV positive (HIV/HCV negative), with an HBV viral load of 8.92 log copies/ml and an HBsAg signal to noise cut-off (S/CO) of 521.62. The full genome sequence of HBV from this patient was assembled and classified as genotype A.After sequence-based characterization of HIV, HBV, and HCV in a large viral surveillance study conducted among 10,457 participants in Kinshasa, the DRC, from 2017 to 2019, a subset of 860 specimens were selected to be screened for viral pathogen discovery by mNGS. The subset included specimens testing negative for all three viruses and those that were singly or doubly infected . mNGS scOrthobunyavirus genus. The genome was assembled de novo, resulting in 94 per cent (706/13.7X), 96.2 per cent (562/17.3X), and 92 per cent (169/29.9X) coverage (reads/depth) for the L, M, and S segments, respectively. To rule out cross-contamination and confirm this discovery, an untouched aliquot of the original specimen was processed at a separate location (Abbott) using alternate extraction and library preparation methods and was successfully detected again by mNGS. For all three segments, the top BLAST hit was Nyangole virus, which was identified in 2019 (just prior to our discovery) in a febrile pediatric patient from Tororo, Uganda, diagnosed with malaria and pneumonia  infections.We developed a research-use-only quantitative PCR assay based upon the index patient’s viral sequence for dual detection of the L  and S  segments with the goal of identifying additional viremic cases, comprehending genetic diversity, and assessing regional prevalence. Dose-dependent detection of opies/ml . For RdRDakHB 754) appeared a near-identical match to the CHRB594 strain we identified in Kinshasa. At the protein level, amino acid identities for L, M, and S segments were 99.1 per cent , 98.7 per cent , and 99.5 per cent (214/215), respectively  were silent, and only eighteen were non-synonymous. There were 113/4,623 (2.44 per cent) nucleotide mismatches (106 transitions and 7 transversions) in the M segment of CHRB594 compared to Bangui DakHB 754. Again, most mutations  were silent, with only eighteen non-synonymous mutations. Finally, for S, there were 22/1,023 (2.15 per cent) nucleotide mismatches (22 transitions and 0 transversions) compared to Bangui DakHB 754. Every mutation in the nucleocapsid open reading frame except one was silent. Thus, we identified a second strain of Bangui virus nearly 50 years after the original isolate.In March 2021, the US Centers for Disease Control recovered the genomes of thirty-five previously un-sequenced bunyaviruses in their Arbovirus Reference Collection, isolated from vertebrates and arthropods over 66 years and from 27 countries . Most vi in mice . While tAn ML tree reconstruction using amino acid sequences available for 721 orthobunyavirus isolates showed a well-supported clustering pattern for the L segment of Bangui virus with Nyangole virus in a serologically unclassified clade  . The BanTo gain resolution for the clustering and branching distances between the viral species closest to Bangui virus, we focused on twenty-two viruses in the Tanga and Anopheles A/B serogroups , inset, We also observed genomic reassortment for some viral species within this group as a potential source of genetic diversity. For example, the M segment of Anopheles A virus branched most closely to Las Maloyas virus, whereas in the L and S segments, it branched most closely to Lukuni virus . AdditioTo further explore the evolutionary history of both Bangui virus strains within the context of the Tanga and Anopheles A/B serogroups, molecular clock analyses were performed. Root-to-tip distance regression of the ML trees using TempEst and TreeTime failed to establish a molecular clock signal . BETS usDue to the lack of temporal signal obtained from the Tanga/Anopheles groups in isolation, we addressed the inference of evolutionary history by calibrating the molecular clock using the closely related California serogroup , for whiWith this timescale established, the ancestor of the Bangui–Nyangole clade’s M segment diverged with a median time of ∼14,000 years ago . The emedS), non-synonymous substitution rates (dN), and the ratio between them . These values were assessed both between sites and between branches from the estimated temporal topologies.Two fundamental questions arose when considering the emergence of Bangui virus to be ∼9,500 years ago: (1) why has Bangui virus gone unnoticed for nearly 50 years? and (2) why does it appear to not actively circulate in the human population? Bangui virus and its close relatives Nyangole, Tataguine, and Tacaiuma have been documented to induce acute fever, rash, and arthralgia in humans . AdditiodS); however, most of those mutations did not generate changes in the corresponding amino acid residues (dN). Hence, all values of ω (dN/dS) were lower than 0.25 (ω < 1).Based on the Bayesian posterior probabilities determined by the M2 and M8 site models, no codon sites in the L, M, and S segments were found to be under positive selection pressure . For allω at each bifurcation in a non-time-stamped Bayesian MCC tree for each segment, again using the branch-site models implemented in CODEML. We observed that the earliest diversification events in ancestors of the entire Tanga/Anopheles group carrying the L segment were driven by positive selection; however, none of the subsequent diversification in L appears to be  and mNGS (n = 859) were unsuccessful, yet not altogether surprising given the results of our phylogenetic analysis. Of the >170 viruses in the Orthobunyavirus genus, only ∼30 are known to cause diseases in mammals and result in outbreaks, such as LaCrosse, Oropouche, and Schmallenberg viruses    or the Je (RNAi) . Thus, dles spp. , the RNAles spp. , left.It has been observed experimentally that some members of the Anopheles serogroups can suppress host interferon (IFN)-β response  althoughMed8 (The pervasive selection also acts on Bangui virus as a purifying force limiting the transmission and circulation in the incidental human host , right. Med8 , thus alMed8 . While tMed8 .East and Central African nations have a rich history of Orthobunyaviral isolation and identification by non-sequencing techniques since at least 1930. Isolated or repeated Batai, Bunyamwera, Bwamba, Germiston, Ilesha, Ngari, Nyando, and Pongola virus outbreaks have been documented in Central African Republic, Cameroon, Uganda, Kenya, Tanzania, Sudan, and Somalia . Thus, tNot all zoonotic viruses warrant the same level of attention. Here, we show that the genetic composition of a Bangui virus strain isolated 50 years ago is essentially the same as the one collected in the present day; coupling the lack of anti-host defenses, the dearth of variant strains, and the absence of recent positive selection events, we believe that Bangui virus and others related to it are hindered from causing active outbreaks in the human population. The phylogenetic approaches shown here may provide the tools to predict whether a virus is of little concern or represents an emerging threat. This has a great value from the standpoint of public health and whether to invest in developing targeted molecular and serologic assays for the virus. As metagenomic approaches lead to the discovery and characterization of an increasingly large number of new viruses, applying analyses like those described here will be essential to inform which viruses merit such concern.vead018_SuppClick here for additional data file.


Nicotiana benthamiana, then investigated the effects of these different co-infections on tlaRNA systemic movement within the host, and on virus accumulation, and aphid and mechanical transmission of each of these viruses. We found that CMoV alone could support systemic movement of each of the tlaRNAs, making this the second report to demonstrate such an interaction between an umbravirus and tlaRNAs. We also report for the first time that CMoV could also impart mechanical transmissibility to the tlaRNAs sigma and ST9, and that co-infections of either of these tlaRNAs with both TuYV and CMoV increased the efficiency with which TuYV could be mechanically co-transmitted with CMoV.Mixed infections of a plant infecting polerovirus, umbravirus, and/or tombusvirus-like associated RNAs (tlaRNAs) produce unique virus disease complexes that exemplify “helper-dependence” interactions, a type of viral synergism that occurs when a “dependent” virus that lacks genes encoding for certain protein products necessary for it to complete its infection cycle can utilize complementary proteins encoded by a co-infecting “helper” virus. While much research has focused on polerovirus-umbravirus or polerovirus-tlaRNA interactions, only recently have umbravirus-tlaRNA interactions begun to be explored. To expand on the limited understanding of umbravirus-tlaRNA interactions in such disease complexes, we established various co-infection pairings of the polerovirus turnip yellows virus (TuYV), the umbravirus carrot mottle virus (CMoV), and three different tlaRNAs—carrot red leaf virus aRNAs (CRLVaRNAs) gamma and sigma, and the TuYVaRNA ST9—in the model plant  Helper-dependence, also known as transcomplementation, is a particularly interesting example of virus synergism between co-infecting viruses. In such infections, the “helper” virus encodes a gene(s) product that the “dependent” virus lacks but can utilize, and in some instances this interaction is obligatory for the dependent virus to complete its infection cycle as these proteins facilitate within host movement and/or transmission between hosts . In manyMultiple disease complexes comprised of a polerovirus, an umbravirus, and/or a satellite virus or tlaRNA have been identified that are responsible for causing severe disease outbreaks in economically important crops . The groPolerovirus (family Solemoviridae) are phloem limited and obligately vectored by aphids, often in a species-specific manner; they encode their own capsid proteins (CP) and are capable of in planta systemic movement ] plants, which produces significantly enhanced symptom development and TuYV accumulation, and in which the ST9aRNA gains systemic movement and aphid transmissibility through encapsidation by TuYV capsid proteins (One such virus complex is the polerovirus TuYV with the ST9aRNA in shepherd’s purse , and by far the most dramatic increase (172.66-fold) was observed in the NILs of TuYV + CMoV + ST9 infected plants. All other co-infection treatments resulted in non-significant changes in TuYV accumulation . CMoV acGamma accumulation levels increased significantly in the ILs of TuYV + Gamma, CMoV + Gamma, and TuYV + CMoV + Gamma inoculated plants by 3.00-, 25.35-, and 37.25-fold, respectively. Interestingly in the NILs of CMoV + Gamma and TuYV + CMoV + Gamma, Gamma accumulation levels  did not vary significantly from that in the ILs of plants inoculated with Gamma alone . Among tN. benthamiana plants were inoculated using tissue from plants infected with any of the viruses in this study not known to be independently mechanically transmissible (TuYV and tlaRNAs), systemic infections were not observed by any of these viruses; when the rub-inoculated leaves were tested for these viruses, low level amplification of all but Gamma could be detected. This low level detection may simply be attributed to residual inoculum, or it may suggest that some cells could become infected with these viruses by rub inoculation but that the viruses could not move beyond the inoculated cells (data not shown); these results coincide with those from early studies on ST9 in rub inoculated C. b-p leaves  could be detected in aphid inoculated plants, when tissue from plants infected singly or in combination with any of these viruses was used as the inoculum. Co-infection with TuYV did facilitate aphid transmission of CMoV when plants co-infected with TuYV + CMoV, TuYV + CMoV + Gamma, and TuYV + CMoV + ST9 were used as the inoculum source—CMoV was transmitted to 42%, 43%, and 64% of recipient plants, respectively, and TuYV was, respectively, transmitted to 100%, 77%, and 77% of recipient plants. Neither Gamma nor Sigma became aphid transmissible when co-infected with TuYV, despite TuYV being transmitted to 100% and 92% of recipient plants from these inoculum sources. Triple infections of each of these tlaRNAs with TuYV and CMoV did not yield different results, despite TuYV transmission efficiency remaining high . Conversely ST9 was successfully aphid transmitted with low efficiency (10%) to a single recipient plant from plants co-infected with TuYV + ST9, and was aphid transmitted to 7% of recipient plants when the inoculum source came from plants also infected with TuYV + CMoV. Transmission efficiencies of TuYV in these treatments were 100% and 77%, respectively. These results are summarized in While there are many studies on polerovirus-tlaRNA and polerovirus-umbravirus interactions in disease complexes harboring various combinations of these viruses, there exist only two recent studies that have begun to touch upon umbravirus-tlaRNA interactions . While wN. benthamiana plants, the most interesting result we found was that almost all co-infections that included CMoV (with the exception of CMoV + Gamma) induced enhanced symptom development in inoculated plants relative to those infected with any virus alone or plants co-infected with TuYV and any tlaRNA. Additionally, there were noticeable differences in symptom presentation depending on if CMoV was co-infected with TuYV  or with either Sigma or ST9 (mosaic symptoms on leaves). There were also notable differences in the severity of leaf mosaic symptoms between co-infections that included Sigma (less severe) and those that included ST9 (more severe), both in double infections with CMoV and in triple infections with TuYV and CMoV. These results suggest that, in this model host and disease complex system, CMoV is the key driver of symptom development since symptoms only occurred in co-infections in which it was present. These results also demonstrate marked differences in symptom development with respect to each of the tlaRNAs; as more of these tlaRNAs are being regularly discovered, it will be interesting to further uncover the various ways they differ in the effects they have on symptom development and interactions they have with co-infecting viruses in these unique disease complexes.With respect to symptom development in Cryptotaenia canadensis subsp. Japonica), only CMoV and the CRLVaRNA could be detected in the recipient plant, making this the first reported evidence that an umbravirus may support tlaRNA systemic movement in the absence of a co-infecting polerovirus, although these results could also have other potential explanations. In another recent study by Until recently, it was thought that only poleroviruses were responsible for systemically trafficking tlaRNAs in these disease complexes. However, a recent study by N. benthamiana plants, ST9 only moved systemically 38% of the time, which was odd as these viruses are known to naturally co-occur and form a strong helper-dependence relationship in Capsella bursa-pastoris plants . However, when TuYV was co-inoculated with CMoV and either Sigma or ST9, the efficiency of TuYV mechanical transmission greatly increased. The observed increase in TuYV accumulation in these plants may partially explain the increased rate of mechanical transmission of TuYV. Conversely, the low accumulation of Gamma may help to explain why this tlaRNA could not be mechanically co-transmitted with CMoV.M. persicae aphids when co-infected with either of the poleroviruses potato leaf roll virus (PLRV) or beet western yellows virus (BWYV), along with other examples of compatible interactions between non-naturally co-occurring poleroviruses and umbraviruses, which could have important epidemiological implications for the development of novel disease complexes or transmission of umbraviruses to novel hosts (We also found that CMoV became aphid transmissible when co-infected with TuYV, except from plants co-infected with TuYV + CMoV + Sigma. This supports similar findings that demonstrate CMoV could be transmitted by el hosts . CMoV waTogether, these findings add to the growing body of knowledge on disease complexes involving these types of viruses and provide novel insights into the virus-virus interactions that occur. Such information could potentially be employed in programs aimed at preventing or controlling outbreaks of such disease complexes and perhaps could be used in the development of novel virus-based gene delivery systems.The original contributions presented in the study are included in the article/AE designed and executed the experiments, analyzed the data, and wrote the manuscript. BF reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.


Determination of the size, density, and mass of viral particles can provide valuable information to support process and formulation studies in clinical development. Analytical ultracentrifugation (AUC), as a first principal method, has been shown to be a beneficial tool for the characterization of the non-enveloped adeno associated virus (AAV). Here, we demonstrate the suitability of AUC for the challenging characterization of a representative for enveloped viruses, which usually are expected to exhibit higher dispersity than non-enveloped viruses. Specifically, the vesicular stomatitis virus (VSV)-based oncolytic virus VSV-GP was used to evaluate potential occurrence of non-ideal sedimentation by testing different rotor speeds and loading concentrations. The partial specific volume was determined via density gradients and density contrast experiments. Additionally, nanoparticle tracking analysis (NTA) was used to determine the hydrodynamic diameter of VSV-GP particles to calculate their molecular weight via the Svedberg equation. Overall, this study demonstrates the applicability of AUC and NTA for the characterization of size, density, and molar mass of an enveloped virus, namely VSV-GP. A fast-growing number of virus-based therapeutics are entering clinical development, such as adeno-associated virus (AAV) for gene therapy and vesicular stomatitis virus (VSV) for both oncolytic cancer therapy . Once D and s are known, the Svedberg equation can be used to determine the particle’s molar mass Svedberg :4\documeOverall, the sedimentation coefficient and the diffusion coefficient indicate the potential of analytical ultracentrifugation to provide insights into hydrodynamic, thermodynamic and density properties of virus particles depending on the experimental approach.Another type of experiment, which would rather be considered a sedimentation equilibrium experiment . Density and viscosity were calculated using SEDNTERP  of VSV was replaced by the glycoprotein (GP) of lymphocytic choriomeningitis virus, was used  the ZetaViewRP Philo ) was takThe wildtype of VSV was reported to be anisotropic  cumulative sedimentation coefficient distributions in Fig. However, already with the results from NTA and AUC available, the molar mass of VSV-GP was estimated according to Eq. Within this study, AUC was used to characterize the oncolytic virus VSV-GP. Although, the fractionating method of AUC offers the opportunity to investigate dispersity in samples, limitations of the method occur due to the multidimensionality and complexity of enveloped virus particles. As the sedimentation coefficient depends on geometry, mass, and density, several different AUC experiments that aim at isolating individual disperse properties should be conducted. Especially, for large viral particles, other techniques that provide additional information, like nanoparticle tracking analysis, can help in providing more information on the quality of viral therapeutics. Overall, AUC can be a valuable tool to give information on disperse properties such as size, density, and mass, to support process and formulation development of viral therapeutics.


We provide the first empirical evidence on the direct link between locus of control and self-control, and how they interact in explaining a range of health outcomes. Using rich Australian survey data, we find that, while the two traits are distinct constructs, a greater internal locus of control is associated with higher self-control. The association between locus of control and health is reduced once we control for self-control, suggesting that self-control mediates at least part of this relationship. Finally, an internal locus of control amplifies the beneficial effects of self-control particularly for physical health. •We study the link between locus of control, self-control, and health outcomes.•A greater internal locus of control is associated with higher self-control.•Self-control mediates part of the relationship between locus of control and health.•An internal locus of control strengthens the beneficial effects of self-control. For exaThis paper investigates the joint role for people's health of locus of control and self-control—two personality traits that independently have been shown to be key for understanding people's health and health behavior. Locus of control describes the belief about the extent to which life events are due to own actions—characterizing people with an internal locus of control—rather than outside forces beyond one's control—characterizing people with an external locus of control . There iSelf-control, in turn, is often described as the ability to override automatic impulses . It helpempirical evidence on the link between locus of control and self-control is surprisingly limited within the broader psychology and economics literature. Thus, while locus of control and self-control are distinct concepts , their iWe make several important contributions to this literature. First, to our knowledge, this study is the first to empirically test the theorized association between locus of control and self-control in a population representative survey. The Household, Income and Labour Dynamics in Australia (HILDA) survey—as the first population representative sample to include both concepts—provides measures of both locus of control and self-control in its 2019 wave. Our empirical results indeed support the theory that a more internal locus of control is related to greater self-control. At the same time, however, we demonstrate empirically that locus of control and self-control are distinct concepts. This insight invalidates the approach taken in some studies of interpreting measures of locus of control to capture self-control or vice versa. For example, when studying the relationship between self-control and obesity, Second, we investigate whether self-control is a channel through which locus of control affects people's physical and psychological wellbeing. Finally, we investigate how locus of control and self-control—both having been shown to independently impact health—interact in predicting people's health. We show that, while having high self-control is related to better health, having an internal locus of control amplifies the beneficial health impacts of self-control, particularly for physical health. These findings suggest that there could be crucial benefits for policymakers of targeting both locus of control and self-control simultaneously when addressing population health: Policies to enhance self-control for the benefit of improving individual health outcomes may likely be of limited success among those with an external locus of control. Conversely, intervention programs designed to improve both people's self-control and their locus of control could yield great efficiency gains.The remainder of the paper is organized as follows: Section 2Locus of control is the belief about whether life events are due to own actions  or due to outside forces beyond your control  , while sWhile being distinct constructs, locus of control and self-control are, however, conceptually related. At the same time, the effect of self-control on people's health behaviors—and thereby health outcomes—could also depend on their locus of control. In the next sections, we conduct an empirical investigation into each of these hypothesized relationships individually. Our analyses are descriptive rather than causal; we therefore rely on the theoretical considerations outlined in this section to guide our interpretation of the direction of effects. For example, we do not consider the reverse relationship in which locus of control could mediate the effect of self-control on people's health. Similarly, as it aligns with theory, we interpret any joint effects of locus of control and self-control in predicting people's health as support for locus of control acting as a moderator, even though empirically either could of course be the moderator in the interaction.3We base our empirical analyses on data from the Household, Income and Labour Dynamics in Australia (HILDA) survey. HILDA is a nationally representative household panel, started in 2001 and surveying more than 17,000 Australians annually. The survey provides rich information on a broad range of people's life aspects, including, for example, their socioeconomic conditions, labor market history, relationships, and health and wellbeing. Importantly, in its 2019 wave HILDA includes measures of both locus of control and self-control as part of the Self-Completion Questionnaire (SCQ), making it the ideal dataset for the purpose of this study.Measuring locus of control and self-control. In HILDA, locus of control is elicited through seven items from the HILDA includes the Brief Self-Control Scale  are measured contemporaneously; our empirical analysis is thus descriptive in nature. As such, our empirical evidence provides an understanding of population-wide patterns in the interrelationship between locus of control, self-control, and people's health. While not causal, such descriptive evidence can be valuable guidance for generating hypotheses, understanding mechanisms, and interpreting findings, and provide a target for practitioners and researchers to focus their efforts on . In addiThis stability of both traits goes a long way in addressing potential concerns of endogeneity, as it reduces the risk of a spurious correlation  or reverse causality in our analyses focusing on health outcomes . Nonetheless, other sources of endogeneity may still be present, making our empirical analysis descriptive in nature. Also, as both are self-reported, measurement error may plague the locus of control and self-control scores. For locus of control we have repeated measurements, such that, in additional robustness checks, we can address potential measurement error in two ways: First, we test the sensitivity of our results to constructing locus of control as the average across all waves with the trait available for each individual rather than just using the 2019 observation. The HILDA survey has included the Health outcomes. HILDA surveys people's physical and mental wellbeing in detail, which allows us to study a wide range of aspects of people's health including (i) their overall and physical health; (ii) health behaviors; and (iii) their mental health and wellbeing. We capture overall and physical health through people's self-rating of their health in general on a scale from 1 (poor) to 5 (excellent), as well as two measures derived from the SF-36 Health Questionnaire: the Physical Component Summary (PCS), (standardized to mean zero and standard deviation one) and the general health subscale . In addition, we use information on people's Body Mass Index (BMI) to construct indicators for being overweight (BMI ≥25) and for being obese (BMI ≥30).Health behaviors include whether people are inactive, i.e., do not at all participate in physical activity for at least 30 min, whether they currently smoke and, if so, the number of cigarettes per week, whether they drink alcohol at least once a week, and whether they have three or more alcoholic drinks per occasion. Some of these behaviors can be directly related to the other health outcomes, e.g., inactivity may lead to being overweight. Therefore, in addition to being outcomes worth studying, they likely further serve as potential mechanisms for (some of) the other health outcomes.Finally, we measure people's mental health and wellbeing in various ways. We rely on the SF-36 for measures of the Mental Component Summary (MCS) score (standardized to mean zero and standard deviation one) and the Mental Health subscale . In addition, we calculate an indicator for psychological distress that equals one if respondents' Kessler-10 score is greater or equal to 30, indicating high or very high distress see . To measA full list of all variables and their definitions is provided in Sample. We base our analyses on data from the 2019 wave of HILDA, which includes 17,462 respondents, of whom 16,150 completed the SCQ. Of these potential observations, we exclude 852 respondents with missing information on either locus of control or self-control. We exclude a further 10 observations with incomplete information on the set of control variables that we include in our analyses: age, gender, education, immigrant status, Indigenous status, and state of residence. Our final analysis sample thus consists of 15,288 respondents. In 4We start our empirical analysis by investigating the link between locus of control and self-control. An exploratory factor analysis of the total of 20 items from both the locus of control scale and the BSCS reveals two factors with an eigenvalue above one. Restricting the factor analysis to two factors in a next step, we report the resulting factor loadings with oblique rotation in i, SCi is their self-control score, LOCi is their locus of control score, Xi is a vector of control variables, and Given that self-control and locus of control are distinct concepts, we next consider the question of whether, and to what extent, locus of control predicts self-control to investigate their relationship. We estimate an OLS regression equation of the form:We focus on this parsimonious set of control variables as they are arguably exogenous, except for education and state of residence. While state of residence is a choice, it is unlikely to be systematically linked to locus of control or self-control. The completion of education, in contrast, is likely affected by both. We include it nonetheless, as we are interested in capturing the contemporaneous link between the two that operates beyond education, which most people in our sample have completed (long) in the past. We refrain from including other personality traits in any of our analyses, such as the Big Five, as they are conceptually related and would thus blur the interpretation of our result (see The locus of control parameter in column (1) denotes the unconditional correlation coefficient with self-control. The value suggests a positive correlation between locus of control and self-control of 0.357. This correlation is very close to the range between 0.37 and 0.40 found in other studies . Adding Overall, our results suggest that a one standard deviation increase in  locus of control is associated with an increase in self-control by about 0.36–0.37 standard deviations, on average. Thus, in line with our expectations, a more internal locus of control is correlated with greater self-control. This finding implies that self-control could potentially be a mechanism through which locus of control improves people's life outcomes , which w5Next, we investigate whether self-control—given it is distinct from but related to locus of control—is potentially a mechanism through which locus of control improves people's health outcomes. We focus on physical health, health behaviors, and mental health, which have independently been linked to both locus of control and self-control. Specifically, we examine what proportion of the association between locus of control and the respective health outcomes can be explained by self-control.Hi is the health outcome of interest and all other variables are defined as before. We then add self-control to the model and estimate:For this purpose, we first estimate models of the form:Hi we first regress the outcome on locus of control and a set of controls, after which we also add self-control in a subsequent model. Our focus here is on the extent to which the locus of control parameter in equation Thus, for each health outcome To formalize the mediating relationships further, we estimate a mediation model similar in approach to that of The main results are reported in Once we add self-control to the model, we find that—as expected—greater self-control is significantly related to improved physical health, better mental health and wellbeing, and lower likelihood of unhealthy behaviors  effect in There are only two exceptions—physical inactivity and alcohol consumption. There is no evidence that self-control mediates locus of control's relationship with physical inactivity. In contrast to all other outcomes, for physical inactivity there is no significant association with self-control, and the Overall, consistent with These findings also hold true when we additionally control for the Big Five personality traits  that are elicited using a 36-item inventory in 2017 see . Despite6The results in the previous section show that, for most outcomes, having greater self-control is associated with better health, on average. A more internal locus of control tends to be associated with better health outcomes as well. In this section, we test whether having an internal locus of control amplifies the beneficial health effects of having more self-control, in line with our theoretical considerations.ILOCi equals 1 if a respondent has a locus of control score above the 25th percentile, and 0 otherwise. We use this binary measure for two reasons: First, this allows us to capture a non-linear effect, in which effects differ for people with a pronounced external locus of control compared to others—who, for simplicity, we refer to as having an internal  locus of control, even though the level of internality may be quite heterogeneous within this group. Second, the binary measure eases interpretation of the interaction term. The threshold is, however, a somewhat arbitrary choice. Therefore, while we choose the 25th percentile as the relevant cutoff in our main estimations, we employ different thresholds such as the median or the 10th percentile in robustness checks (see below).For this purpose, we estimate models of the form:Our parameter of interest is Again, these findings are robust to including the Big Five personality traits as additional control variables , to charOverall, our findings thus suggest that an internal locus of control has the potential to amplify the health benefits of greater self-control—albeit not equally across all types of health outcomes. It is particularly true for all physical health outcomes, but also for physical inactivity and health satisfaction.7Locus of control and self-control are key personality traits for understanding people's health and health behavior. An internal locus of control and greater self-control have each been linked, for example, to a healthier lifestyle and greater physical and mental wellbeing . The findings and views reported in this paper, however, are those of the authors and should not be attributed to the Australian Government, DSS, or any of DSS’ contractors or partners. DOI: 10.26193/YP7MNU.Ferdi Botha: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Writing – original draft, Writing – review & editing. Sarah C. Dahmann: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Writing – original draft, Writing – review & editing.Declarations of interests: None.