A single amino acid change at position 226 in the hemagglutinin (HA) from glutamine (Q) to leucine (L) has been shown to play a key role in receptor specificity switching in various influenza virus HA subtypes, including H9. We tested the flexibility of amino acid usage and determined the effects of such changes. The results reveal that amino acids other than L226 and Q226 are well tolerated and that some amino acids allow for the recognition of both avian and human influenza virus receptors in the absence of other changes. Our results can inform better avian influenza virus surveillance efforts as well as contribute to rational vaccine design and improve structural molecular dynamics algorithms. in vivo replication advantage. This study shows the flexibility of position 226 of the HA of H9 influenza viruses and the resulting effect of single amino acid changes on the phenotype of variants in vivo and in vitro.Influenza A viruses (IAVs) remain a significant public health threat, causing more than 300,000 hospitalizations in the United States during the 2015–2016 season alone. While only a few IAVs of avian origin have been associated with human infections, the ability of these viruses to cause zoonotic infections further increases the public health risk of influenza. Of these, H9N2 viruses in Asia are of particular importance as they have contributed internal gene segments to other emerging zoonotic IAVs. Notably, recent H9N2 viruses have acquired molecular markers that allow for a transition from avian-like to human-like terminal sialic acid (SA) receptor recognition via a single amino acid change at position 226 (H3 numbering), from glutamine (Q226) to leucine (L226), within the hemagglutinin (HA) receptor-binding site (RBS). We sought to determine the plasticity of amino acid 226 and the biological effects of alternative amino acids on variant viruses. We created a library of viruses with the potential of having any of the 20 amino acids at position 226 on a prototypic H9 HA subtype IAV. We isolated H9 viruses that carried naturally occurring amino acids, variants found in other subtypes, and variants not found in any subtype at position 226. Fitness studies in quails revealed that some natural amino acids conferred an IMPORTANCE A single amino acid change at position 226 in the hemagglutinin (HA) from glutamine (Q) to leucine (L) has been shown to play a key role in receptor specificity switching in various influenza virus HA subtypes, including H9. We tested the flexibility of amino acid usage and determined the effects of such changes. The results reveal that amino acids other than L226 and Q226 are well tolerated and that some amino acids allow for the recognition of both avian and human influenza virus receptors in the absence of other changes. Our results can inform better avian influenza virus surveillance efforts as well as contribute to rational vaccine design and improve structural molecular dynamics algorithms. N-acetylneuraminic acid , although horse-adapted IAV strains develop a preference for N-glycolylneuraminic acid (––The hemagglutinin (HA) of influenza A viruses (IAVs) plays a central role in the viral life cycle through its involvement in receptor recognition, virus attachment, membrane fusion, and virus entry . The HA 2–nic acid . In natunic acid . It is wnic acid –9. Throuic acid –. Seminalic acid –, 12. Theic acid –. In contic acid –, 15. Theic acid ––18.In vivo, however, strong selection toward L226 was observed in quail, highlighting the limitations of in vitro systems to fully recapitulate virus-host interactions in natural hosts. The results provide new insights into the biology of H9N2 influenza viruses and potential avenues for development of live attenuated virus vaccines against the H9 and other influenza virus subtypes.H9N2 viruses are endemic in most of Asia, the Middle East, and parts of Africa, where they have caused disease outbreaks in chickens, quail, and other minor poultry species . H9N2 vi18–25–nnn226H9N2 and equi226H9N2 virus libraries) (nnn226H9N1 and equi226H9N1 virus libraries)  . We alsobraries) . The twonnn226H9N2 library revealed asparagine  and alanine , which have not yet been found in natural isolates, to be the most abundant. Other naturally occurring amino acids included histidine , isoleucine , serine , and methionine   cells, virus clones were analyzed by partial sequencing of the HA gene segment to identify the codon encoding amino acid 226. For simplicity, we refer to variant amino acids (or viruses) as those that carry an amino acid different from L226. Throughout the article we make the distinction of whether a variant amino acid has been found in natural isolates or not. Analysis of 12 individual virus clones from the , n = 1) . Interesequi226H9N2 virus library revealed that N226 (n = 8) was the most commonly selected amino acid, as was seen with clones obtained from the nnn226H9N2 virus library , I226 (n = 1), M226 (n = 1), and S226 (n = 1). In addition, clone selection from the equi226H9N2 virus library resulted in viruses with threonine , phenylalanine , cysteine , glycine , and valine . Once again, L226 and Q226 viruses were absent after clonal selection of the equi226H9N2 virus library. The absence of these two amino acids is not due to a misrepresentation of the corresponding codons in the preparation of the virus library  or a defect in virus rescue. Both of these viruses were independently recovered in the H9N2 background using PCR reverse genetics and the corresponding primer set, indicating that they are viable, as expected (n = 17) and the naturally occurring but rare variant H226 (n = 13) were the most frequently obtained among the virus clones. Noteworthy, the naturally occurring L226 (n = 12) was the most favored amino acid in HA when paired with the N1 NA , we proceeded with an alternative approach to give each amino acid the same representation in the library. Analysis of 24 individual virus clones produced from the  library . Also coexpected . Intereshe N1 NA  but not To investigate receptor-binding activity, we carried out HA assays using chicken red blood cells (RBCs) treated with various concentrations of neuraminidase. As shown in equi226H9N2 and equi226H9N1 virus libraries. We assessed the ability of the virus clones to agglutinate red blood cells from different species using stocks grown in MDCK cells, as previously described , most likely due to its low RBS binding activity.Amino acid changes around the receptor-binding site are known to contribute to the antigenic drift of the resulting viruses , 33. TheP < 0.0001), M226 (P < 0.05), and V226 (P < 0.05) viruses, which that grew to significantly lower titers, while the Q226 virus (P < 0.0001) grew to higher titers than the L226 virus . The I226 virus replicated at consistently lower titers during the course of the experiments (P < 0.0001) both at 48 and at 72 hpi. In DF1 cells, the pattern of growth of H9N2 variant viruses was similar to the growth kinetics in MDCK cells, the I226 virus replicated to lower titers at every time point except at 24 hpi, and the Q226 virus replicated to higher titers than the other variants tested . The S226 variant virus grew to titers similar to those of the I226 virus at 72 hpi, while there was no difference between the L226 virus and the variant viruses at 72 hpi. In MDCK cells and on the PR8 backbone, all H9N1 viruses replicated to lower titers than wild-type PR8 (H1N1) virus (P < 0.0001), N226 (P < 0.001), and F226, S226, and H226 (P < 0.05) viruses, which grew to lower titers in MDCK cells. Interestingly, in DF1 cells, all H9N1 viruses replicated to similar titers and to titers significantly higher (∼2 log10) than the titer of the PR8 virus, suggesting that the H9 HA was responsible for improving virus replication in chicken cells  of 0.01, and supernatants were collected at different times postinfection. Variant viruses exhibited diverse replication profiles in MDCK and DF1 cells compared to the L226 virus . At 24 h26 virus . At 72 hs tested . By 72 h1) virus . At 48 hen cells . OverallWe analyzed α2-3SA and α2-6SA receptor specificity using two independent assays: we performed glycan binding assays using a custom glycan microarray with linear, O-linked, and N-linked sialosides  and soliThe pattern of influenza virus attachment (PVA) in the respiratory tract is an indicator of virus tropism, which can determine the efficiency of viral replication and transmission . Since t36–in vivo, we designed a competition study to investigate whether position 226 confers a relative fitness advantage. Thus, we inoculated young quail with mixtures of the mutant viruses on the H9N2 background. Groups of quails were designed based on the presence or absence of the L226 and Q226 viruses in the mixture used for inoculation. Quails were randomly assigned to 4 groups (n = 6/group). The birds in group 1 received an equal mix of the H9N2 viruses without L226 and Q226 (varΔLQ). Group 2 (var+Q) birds received the H9N2 virus mix along with the Q226 virus, group 3 (var+L) birds received a similar mix with the L226 virus, and group 4 (var+LQ) birds received a mix of all the H9N2 variants and both the L226 and Q226 viruses. A fifth group of quail was mock inoculated with PBS as a negative control (not shown). At 2 days postinoculation (dpi), we introduced naive quail (n = 6/group) to monitor transmission. Over the course of 14 days, infected birds displayed minimal signs of disease, as has been previously reported   . Virus tvarΔLQ group showed delayed replication kinetics. While direct-contact quail in other groups shed an average of 106 TCID50 equivalents/ml by 2 days postcontact (dpc), the average virus titer in tracheal swabs of varΔLQ direct-contact birds was significantly less than that for all other contact groups (P < 0.001) (varΔLQ group was significantly higher than that shed by birds in the other groups (P < 0.05). At 5 dpi and 6 dpc, 3 birds from each group were sacrificed for virus titration of the lungs and trachea. Viral titers were higher in the upper respiratory tract than in the lower respiratory tract in both inoculated and contact quails (Direct-contact quail in the < 0.001) . By 6 dpt quails . A trendn = 6/group) and 4 and 8 dpc  were subjected to whole-genome sequencing by NGS using the Illumina MiSeq platform. By 3 and 5 dpi, birds in the var+LQ group shed predominantly the L226 variant at a frequency of between 51 and 96% by 3 dpi (var+L group shed mainly the L226 variant (43 to 98% frequency). The I226 variant was also identified in one quail at a frequency of 33%. We observed a general increase in the proportion of the other variants being shed in the absence of the L226 variant in both the var+Q and varΔLQ groups. However, the L226 variant was the consensus variant in 3 quails in the var+Q group by 3 dpi, while 2 birds shed the M226 variant, and the I226 variant was found in 1 quail. Without the commonly found L226 and Q226 variants, the M226 variant outcompeted all other variants in the varΔLQ group, being shed at a frequency of between 52 and 84% in inoculated quails at 3 dpi. The V226, F226, and I226 variants were also identified in the varΔLQ group. Though leucine was not a component of the mix in the var+Q and varΔLQ groups, the L226 variant in the var+Q and varΔLQ inoculated groups could have emerged from a single mutation in the second nucleotide of the codon encoding H226, the third nucleotide of the codon encoding F226, or the first nucleotide of the codon encoding either F226, I226, M226, or V226, which were present at lower frequencies in both groups.To determine what amino acids were predominant in inoculated quails and what amino acids were associated with contact transmission events, tracheal swab samples collected at 3 and 5 dpi (by 3 dpi . In one var+L and var+LQ groups shed the L226 variant at 4 and 8 dpc (1 quail in the var+L group shed the M226 variant by 4 dpc)  were identified in the tracheal swabs of contact birds at 4 dpc  ; Fig. 8B viruses ; by 8 dpat 4 dpc . By 8 dpn = 17 out 20) have been associated with the L226 variant (n = 25 out of 45) than to the L226 variant . The differences in susceptibility to the L226 versus Q226 variants in humans versus pigs can perhaps be better explained by their different SA receptor distribution profiles  or that are uncommon  in the background of the H9 HA . Like the L226 variant, the Q226 variant was obtained only when paired with the N1 NA in the PR8 virus background. These results indicate that in vitro and in vivo fitness does not necessarily correlate and question whether some of the variants observed in natural isolates could indeed be artifacts of the method used for virus growth, something that could be easily ascertained by sequencing the virus directly from the field sample.Receptor engagement is a critical step in influenza virus infection and is mediated by the HA. It is well-known that changes to amino acids in the receptor-binding site of HA modulate receptor recognition, which in turn influences host adaptation. Several studies have linked the changes at positions 226 and 228 (H3 numbering) to receptor preference. In the context of H2, H3, and H4 HA subtype viruses, the combination of Q226/G228 allows for avian-like α2-3SA recognition, while the L226/S228 combination favors human α2-6SA recognition , 40, 41. variant . It coulhe H9 HA . It is uUsing cell avidity, solid-phase binding, glycan array analyses, and tissue-binding profiles, we determined the receptor preference of these single-amino-acid variants. In terms of relative α2-3SA and α2-6SA preference, the two ends of the spectrum are represented by the Q226 variant, which binds strongly to sugars containing α2-3SAs, while the L226 variant preferentially binds sugars containing α2-6SAs. These observations are consistent with previous reports , 42. Thein vitro data showed a similar fitness for all of the mutants, as they replicated to similar titers by 72 hpi in avian (except the S226 and I226 variants) and mammalian (except the I226 variant) cell lines. Results from the in vivo competition studies in quails, however, indicate that there are bottlenecks affecting the virus phenotype in vivo that cannot be recapitulated by in vitro systems. Due to limitations in the number of animal studies that could be performed, we decided to ascertain the relative fitness advantage of the position 226 variant viruses in an in vivo competition model. Undoubtedly, the L226 virus showed an in vivo advantage in the quail model. Interestingly, the L226 variant was the most thermostable virus among the H9N2 variants  can lead to the emergence of the L226 codon (CTG). Similarly, the L226 variant detected in birds in the var+Q group could have easily emerged from a single nucleotide change in the Q226 codon (CAA) to produce the L226 codon (CTA). It is noteworthy that the N226 variant was detected at a high frequency in one direct-contact quail by 8 dpc, despite having the lowest frequency (∼4%) of all the identified variants in the varΔLQ group by 3 dpi. Unexpectedly, we found the variant K226, which was not part of the initial inoculum and which represented ∼1.5% of the variants, in 1 contact quail in the var+Q group at 8 dpc. The significance of the N226 and K226 variants for transmission and their potential emergence in nature remain to be determined. Despite generating NGS information for 2 different time points postinfection in both inoculated and direct-contact quails, we should note that our data represent a snapshot of a dynamic replication and transmission event. We cannot exclude the possibility of a more complex mixed population of variants initiating infection/transmission, although our data suggest the progressive dominant effect of one variant over the course of infection. Additional mutations were identified throughout the viral genome depending on the quail sample analyzed (var+L group) is characteristic of recent BJ/94-like H9N2 isolates in China  guidebook of the Office of Laboratory Animal Welfare  and PHS l-glutamine, and 2.5% HEPES . Cells were cultured at 37°C in a humidified incubator under 5% CO2.Madin-Darby canine kidney (MDCK) and human embryonic kidney 293T cells were a kind gift from Robert Webster, St. Jude Children’s Research Hospital, Memphis, TN. Chicken fibroblast-derived (DF1) cells were a kind gift from Siba Samal, University of Maryland, College Park, MD. Cells were maintained in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum , 1% antibiotic-antimycotic , 1% 226HA PCR library was generated by focusing on the codon encoding amino acid 226 using two different approaches. In the first approach, a PCR product was produced from pDPAO1-767 using the primer set t1FragFwd and WF10-HA713-757nnn (primer sequences are available upon request). The resulting nnnHA757-3′5′-t1 PCR product included the t1 terminator sequence and the HA fragment with the NNN codon at position 226. In the second approach, an equivalent PCR product, equiHA757-3′5′-t1, was generated from pDPAO1-767 using the primer t1FragFwd and 50 pmol/μl of a primer mix containing 20 primers designed to introduce every possible amino acid at position 226. The second half of the HA was amplified from pDPAO738-1742 using the set of primers WF10 HA 738-770 and hPol1Rev to generate the PCR product 5′-HA738pol1-3′ that included the second half of the HA followed by the pol 1 promoter sequence. The full-length reverse genetics-ready HA PCR librarie, nnn226H9PCR and equi226H9PCR were obtained by overlapping PCR using either the nnnHA757-3′5′-t1 or the equiHA757-3′5′-t1 PCR fragment, respectively, along with the 5′-HA738pol1-3′ PCR fragment and the primer pair t1FragFwd and hPol1Rev. The 50-μl PCR mixture contained 10 ng of each PCR product, 25 μl of the PCR master mix, 50 pmol/μl of each primer, and 1.5 μl of dimethyl sulfoxide. PCR amplification was done using a Phusion high-fidelity PCR master mix with GC buffer  under the following cycling parameters: 98°C for 30 s; 98°C for 8 s, 56°C for 1 min, and 72°C for 3 min for 30 cycles; and 72°C for 10 min (The full-length cDNA copy of the HA gene segment from A/guinea fowl/Hong Kong/WF10/1999 (H9N2) virus (WF10) was split into two overlapping fragments and cloned into two separate plasmids. The plasmid pDPAO1-767 contains nucleotides 1 to 767 of the H9 HA gene segment preceded by the 5′ end sequence corresponding to the mouse RNA polymerase 1 (pol 1) terminator (t1). The plasmid pDPAO738-1742 contains nucleotides 738 to 1742 of the H9 HA segment immediately upstream of the human RNA polymerase 1 (pol 1) promoter . The 226r 10 min .nnn226H9PCR and equi226H9PCR amplicon libraries, as previously described  (PR8). For transfection, cocultures of MDCK and 293T cells were seeded in each well of a 6-well plate overnight at 37°C. On the following day, 1 μg of each of the 7 plasmids of either WF10 or PR8 and 1 μg of PCR amplicon were mixed with 16 μl of the TransIT-LT1 transfection reagent , and the mixture was incubated for 45 min. After 45 min, the MDCK/293T cells were overlaid with the transfection mixture and incubated at 37°C for 24 h. At 24 h posttransfection (hpt), the transfection mixture was replaced with fresh Opti-MEM I medium  containing 1 μg/ml of tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin  and 1% ATB-ATM. Supernatants containing rescued viruses were collected at 96 hpt.Independent virus rescue experiments were performed with the escribed , 60. To 0 genome . To gene4 cells/well) in a 96-well plate were infected with 8 serial 10-fold dilutions of the rescued virus library in Opti-MEM I medium  containing 1 μg/ml of TPCK-treated trypsin and 1% ATB-ATM. The variant strains were produced starting from the nnn226H9N2 virus library (n = 12), the nnn226H9N1 virus library (n = 12), the equi226H9N2 virus library (n = 24), and the equi226H9N1 virus library (n = 24). After 72 h of incubation at 37°C, virus supernatants were collected from wells infected with the most diluted sample displaying a cytopathic effect (CPE). This process was repeated once, followed by Sanger sequencing to determine the amino acid at position 226 in HA. A third round of limiting dilution was carried out for samples yet to resolve at position 226 after the second limiting dilution. The viruses were further expanded in MDCK cells, the stocks were aliquoted, and the aliquots were stored at −80°C until use. The virus stocks were titrated by use of the 50% tissue culture infectious dose (TCID50) in MDCK cells, and titers were determined by the Reed and Muench method (Single virus variants in the virus libraries were isolated by limiting dilution assays as previously described . Brieflyh method .Coturnix japonica) were infected with 106 TCID50 of WF10 or the M226 virus and boosted at 14 dpi with the respective inactivated virus containing 1:1 (vol/vol) Montanide as an adjuvant. The quails were bled for serum collection at 14 days after the boost, and the antisera collected were used in hemagglutination inhibition (HI) assays. Standard hemagglutination assays were performed using either 1% horse red blood cells (RBCs)  or 0.5% chicken RBCs or 0.5% turkey RBCs  and expressed in hemagglutination units (HAU) as previously described  to remove any unbound virus, MDCK cells were overlaid with 2 ml Opti-MEM I  medium containing ATB-ATM  and 1 μg/ml TPCK-treated trypsin. For infected DF1 cells, TPCK-treated trypsin was replaced with 5% allantoic fluid. At 6, 12, 24, 48, and 72 h postinfection (hpi), the supernatant was collected and stored at −80°C until viral titration in MDCK cells. The viral titer was determined by measurement of the TCIDh method .N-hydroxysuccinimide (NHS) ester-coated glass microarray slide containing six replicates of 128 diverse sialic acid-containing glycans, including terminal sequences, and intact N-linked and O-linked glycans found on mammalian and avian glycoproteins and glycolipids. The virus stock was amplified in MDCK cells, clarified by centrifugation, and inactivated using β-propiolactone at a final concentration of 0.1%. Inactivated virus samples were then purified by ultracentrifugation at 18,000 rpm for 2 h at 4°C, resuspended in PBS–5% glycerol, and stored at −80°C. Whole H9 influenza virus samples were diluted to 256 HAU/50 μl  in PBS containing 3% bovine serum albumin (BSA) and incubated on the array surface for 1 h at room temperature (RT) in a humidity-controlled chamber. After 1 h, the slides were washed in PBS and incubated with a virus-specific anti-H9 mouse monoclonal antibody, AIV-H9-HA 3G8  diluted in PBS, 3% BSA. The slides were washed twice in PBS and in distilled water (dH2O) and then dried prior to detection. Slide scanning to detect bound virus was conducted using an InnoScan-n1100AL  fluorescent microarray scanner. The fluorescent signal intensity was measured using Mapix software , and the mean intensity minus the mean background for 4 replicate spots was calculated. A complete list of the glycans on the array is presented in Table S1 in the supplemental material.Glycan array analysis was performed using an 9-HA 3G8   to remove sialic acids. Following neuraminidase treatment, the RBCs were washed twice with PBS and resuspended to a 1% solution with PBS. Then, 50 μl of 1% RBCs was incubated with 50 μl of virus (8 HAU) at RT for 45 min to determine the hemagglutination titer.The cell binding assay was performed as previously described , 66. Chi2O2). The reactions were stopped with 3% H2SO4 after 30 min, unless otherwise stated. Absorbance readings were obtained at 450 nm using a Victor X3 plate reader . Assays were carried out twice, and viruses were tested in duplicate.Solid-phase direct binding assays using sialylglycopolymers were performed as previously described , 62. BriTracheal tissues from influenza virus-negative 4- to 6-week old Japanese quail were used for virus histochemical studies. Virus histochemistry was performed as previously described using fluorescein isothiocyanate (FITC)-labeled viruses . Brieflyn = 6/group) were used for the in vivo competition study. Quail eggs obtained from the College of Veterinary Medicine, University of Georgia, were hatched at the Poultry Diagnostic and Research Center, University of Georgia. A week before virus inoculation, quails were bled and confirmed to be seronegative for IAV exposure. Quails were inoculated with 1 ml of virus mix (0.25 ml administered via the trachea and nares and 0.5 ml via the cloaca) containing a homogenous mixture of variant viruses with or without L226 or Q226 virus. In group 1 (varΔLQ), birds were inoculated with 106 TCID50 virus mix containing the following 10 variant (var) viruses on the WF10 backbone: I226, S226, T226, M226, H226, N226, F226, V226, C226, and G226 viruses. Group 2 (var+Q) birds received all 10 var viruses and the Q226 virus, while group 3 birds (var+L) were infected with the 10 var viruses and the L226 (WF10) virus. Group 4 (var+LQ) birds were infected with a mix of all 10 var viruses along with the L226 and Q226 viruses (var+LQ). A final group of 6 birds served as the negative control receiving 1 ml PBS through the same routes . At 1 day postinfection (dpi), naive quail (n = 6/group) were introduced as direct contacts to determine transmission. At 5 dpi and 6 dpc, 3 quail from each group of directly inoculated and contact quail, respectively, were randomly selected and sacrificed for virus titration in tissues. Tracheal and cloacal swab specimens were collected daily from each bird until 14 dpi. Swabs were suspended in 1 ml 3.7% brain heart infusion (BHI) medium  containing 10,000 U penicillin, 10 mg streptomycin, and 25 μl amphotericin B and stored at −80°C until use in virus titrations.Four groups of 4- to 5-week-old Japanese quail (pC) values with the amount of virus shed from each bird, as previously described  following the manufacturer’s instructions and eluted in 50 μl of nuclease-free molecular-grade water. A one-step quantitative PCR (qPCR) based on the avian influenza virus matrix gene as a surrogate of virus shedding was carried out using the primer/probe set previously described . The qPCescribed .nnn226HA and equi226HA PCR products prior to transfection and on HA from all var virus stocks. Sequences were generated using specific primers, a BigDye Terminator (v3.1) cycle sequencing kit , and a 3100 genetic analyzer  according to the manufacturer’s instructions. For whole-genome sequencing of viral stock and tracheal swab samples, RNA was extracted using an RNeasy minikit  or a MagNA Pure LC RNA isolation kit . Isolated virus RNA served as the template in a one-step reverse transcriptase PCR for multisegment, whole-genome amplification, as previously described . Subsequently, HA assays were carried out on heat-treated viruses in quadruplicate.http://www.fludb.org (accessed on 18 March 2018). The amino acid frequency at position 226 was then analyzed using the protein sequence variant analysis tool provided by the IRD. Whole-genome sequences were then mapped to the WF10 reference sequence using Geneious (version 10.2.3) software (H9 HA sequences were obtained from the Influenza Research Database (IRD)  through software .post hoc Tukey test. A P value below 0.05 (P < 0.05) was considered significant.Data analyses were performed using GraphPad Prism (version 7) software . For multiple comparisons, either one-way or two-way analysis of variance (ANOVA) was performed, followed by a https://www.ncbi.nlm.nih.gov/sra) under accession numbers SAMN10530904 through SAMN10530939 and BioProject accession number PRJNA508785.The Illumina data are available through NCBI’s Short Read Archive (


Limited evidence exists on the value for money of mHealth information programs in low resource settings.This study sought to model the incremental cost-effectiveness of gradually scaling up text messaging services to pregnant women throughout Gauteng province, South Africa from 2012 to 2017.Data collection occurred as part of a retrospective study in 6 health centers in Gauteng province. Stage-based short message service (SMS) text messages on maternal health were sent to pregnant women twice per week during pregnancy and continued until the infant’s first birthday. Program costs, incremental costs to users, and the health system costs for these women were measured along with changes in the utilization of antenatal care visits and childhood immunizations and compared with those from a control group of pregnant women who received no SMS text messages. Incremental changes in utilization were entered into the Lives Saved Tool and used to forecast lives saved and disability adjusted life years (DALYs) averted by scaling up program activities over 5 years to reach 60% of pregnant women across Gauteng province. Uncertainty was characterized using one-way and probabilistic uncertainty analyses.Five-year program costs were estimated to be US $1.2 million, 17% of which were incurred by costs on program development and 31% on SMS text message delivery costs. Costs to users were US $1.66 to attend clinic-based services, nearly 90% of which was attributed to wages lost. Costs to the health system included provider time costs to register users (US $0.08) and to provide antenatal care (US $4.36) and postnatal care (US $3.08) services. Incremental costs per DALY averted from a societal perspective ranged from US $1985 in the first year of implementation to US $200 in the 5th year. At a willingness-to-pay threshold of US $2000, the project had a 40% probability of being cost-effective in year 1 versus 100% in all years thereafter.Study findings suggest that delivering SMS text messages on maternal health information to pregnant and postpartum women may be a cost-effective strategy for bolstering antenatal care and childhood immunizations, even at very small margins of coverage increases. Primary data obtained prospectively as part of more rigorous study designs are needed to validate modeled results. The use of mobile and wireless technology for health   has the In Zanzibar and Malawi, maternal SMS text messaging initiatives have demonstrated a significant effect on the utilization of health services and health outcomes. In Zanzibar, the Wired Mothers Program provided unidirectional SMS text messaging and direct two-way communication using a free call voucher system to provide reminders for ANC visits; gestational age-specific reproductive, maternal, newborn, and child health (RMNCH) information; and an emergency medical response system. Program activities were associated with an increase in ≥4 ANC visits  . In MalaBeyond these programs, the Mobile Alliance for Maternal Action (MAMA) program has provided stage-based maternal health information using SMS text messaging in Bangladesh, India, Nigeria, and South Africa and has supported content to projects in 54 countries globally. With the exception of formative findings from activities in Bangladesh , evidencThe emergence of data suggesting that stage-based SMS text messages on maternal health information may yield improvements in utilization across the continuum of care, from pregnancy, delivery to postpartum, is promising -6. Yet, To complement efforts to determine the effectiveness of MAMA in South Africa, we modeled the incremental cost-effectiveness of gradually scaling up SMS text messaging services to pregnant women throughout Gauteng province, South Africa from 2012 to 2017. This model-based analysis is anticipated to provide an early estimate of the cost-effectiveness of MAMA and inform future efforts to prospectively monitor costs and consequences of maternal SMS text messaging programs in low- and middle-income countries.th birthday; 25% due to undernutrition, 25% in the first 28 days of life, and >50% outside of the formal health sector  and full immunizations). Despite data limitations, findings from the retrospective case-control study suggested that women exposed to SMS text messages were more likely to attend at least the recommended 4 ANC visits  and complete comprehensive care,  than women not exposed  to generFor this model-based analysis, economic costs were estimated from a societal perspective inclusive of program, health systems, and user costs for a 5-year analytic time horizon (2012-2017). Program costs were defined as the costs required to develop, start-up, and support ongoing implementation. These were captured using an ingredients approach based on program activities, drawing from financial records and informant interviews with project implementing partners , and through observations of health care workers providing routine ANC and PNC services within facilities. Costs were further categorized into capital (costs with a life expectancy of >1 year) and recurrent costs, with the former annualized over the lifetime of the project or life span of the item as appropriate and discounted at 3%. Development and start-up phase costs were viewed as one-time activities and similarly annualized over the lifetime of the project.Incremental costs to the health system sought to capture costs associated with registration and increases in utilization. These included provider time costs to register patients into MAMA, as well as to provide routine clinical services during pregnancy and postpartum, including immunizations. These costs were estimated based on informant interviews, with provider salaries drawn from PayScale.com, an online salary, benefits, and compensation information company, and verified by the human resources department of Wits RHI.Costs to users included all out-of-pocket payments incurred for care or treatment-seeking, including direct costs associated with medical care  as well as costs for transport/treatment-seeking and indirect costs due to wages lost resulting from time spent seeking care or away from income-generating activities. These were measured through patient interviews in intervention and comparison arms and generalized to the sample population as rollout occurred.rd edition, which highlights low-cost high-priority interventions for key regions globally.Costs were adjusted to 2015 USD dollars using consumer price indices . CapitalEstimates of service utilization were used to forecast the costs to the health system associated with registering pregnant women to MAMA as well as treating additional cases . AssuminMean costs to users for attending ANC and PNC were drawn from structured interviews and included food, wages lost, child care, and transport costs . The meaUsing the South Africa’s Gross National Income per capita for 2015 of US$6080 as the threshold, program activities have a 100% probability of being cost-effective. At lower willingness-to-pay thresholds, the probability of MAMA being cost-effective increases over time as the number of users increases along with anticipated health effects.To compliment probabilistic sensitivity analyses, we also conducted one-way sensitivity analyses to identify key drivers of the incremental cost per DALY averted . The leath year of implementation were estimated to be US $3.6 million dollars, 59% of which represent costs borne by users to seek care for ANC and PNC, 30% costs to the health system, and 11% program costs. When considered against a status quo comparator, the incremental annual cost to implement MAMA at 60% coverage is US $1.03 million.Study findings modeling the incremental cost-effectiveness of exposure to SMS text messages during delivery and postpartum on care-seeking and childhood immunizations suggest that the cost per DALY averted ranges from US $1985 in year 1 when only 1% of pregnant women are registered to US $200 in year 5 when 60% of pregnant women are included. Societal costs to implement MAMA in the 5To estimate the health effects of SMS text messaging exposure, we drew from sample data on changes in the utilization of services among MAMA users and nearby non-MAMA-using mother-infant pairs for ANC and childhood immunizations up to 9 months. Less than 100 complete records were available in each study arm. When coupled with the existing high rates of service utilization, it meant that we were not powered to measure changes in the utilizations rates for individual vaccines. However, we were powered to measure observed changes in the utilization for ANC and comprehensive care (defined as ANC 1-4 and full immunizations). Based on available data, 1%-4% increases in immunization rates were observed by vaccine type along with a 14% increase in all 4 ANC visits. The latter finding is consistent with changes in coverage observed in Zanzibar as part of the Wired Mothers Program [In the 2 years since the MAMA program ended, the National Department of Health (NDOH) has developed and rapidly scaled a maternal SMS text messaging program called “MomConnect,” which is based on MAMA but with less specific messages on the prevention of mother-to-child transmission of HIV. Like MAMA, MomConnect registers pregnancies and links expectant mothers to gestation age-specific pregnancy information, while also providing access to a help desk for reporting compliments or complaints on service delivery. Since its inception, MomConnect has grown to become one of the largest mHealth initiatives globally, registering >1 million pregnant women in >95% (3300) of health facilities in South Africa to receive SMS text messages on maternal health information . While dElsewhere, data on the value for money of digital health programs are slowly emerging. However, to our knowledge, this is the first study to provide evidence on the value for money of maternal SMS text messaging programs. To date, a dozen peer-reviewed papers comprise the body of evidence on the value for money of mHealth solutions, including CEAs (6 studies) -26, costFrom the outset, we sought to base this analysis on primary data and focus only on the 2-year analytic time horizon of the program. However, primary data collection efforts were hampered by challenges in patient recruitment and data completeness. While we sought to obtain data on immunizations from the paper-based booklets provided by NDOH to mothers and completed by health workers at the time of service delivery, in practice, significant gaps in the completeness and quality of record keeping meant that a large proportion of interviewed participants were excluded from the final analyses. In shifting to a model-based analysis, we sought not only to more rigorously capture uncertainty but also consider the implications of service delivery at scale. Except for variable costs associated with SMS text message delivery, much of the technological costs associated with the MAMA program activities were fixed, irrespective of the scale of implementation. That said, our findings clearly suggest that greater value for money is attained with increasing scale. We hope that further analyses drawing from primary data of maternal SMS text messaging at scale through MomConnect and other initiatives will confirm this.This is a first of its kind study to provide an evidence-informed model of the value for money of maternal SMS text messaging programs. Study findings suggest that SMS text messages to pregnant and postpartum women are cost-effective, according to the GNI per capita thresholds for South Africa. Cost-effectiveness improves with scale. Further efforts are needed to determine the value for money of maternal SMS text messaging under more robust study designs and in differing settings where technological (network coverage and access to mobile phones), epidemiological, and health systems profiles may differ.


A comprehensive district-strengthening approach to address maternal and newborn health was estimated to cost US$177 per life-year gained in Uganda and $206 per life-year gained in Zambia. The approach represents a very cost-effective health investment compared to GDP per capita. A comprehensive district-strengthening approach to address maternal and newborn health was estimated to cost US$177 per life-year gained in Uganda and $206 per life-year gained in Zambia. The approach represents a very cost-effective health investment compared to GDP per capita. The primary objective of this study was to estimate the costs and the incremental cost-effectiveness of maternal and newborn care associated with the Saving Mothers, Giving Life (SMGL) initiative—a comprehensive district-strengthening approach addressing the 3 delays associated with maternal mortality—in Uganda and Zambia. To assess effectiveness, we used a before-after design comparing facility outcome data from 2012 (before) and 2016 (after). To estimate costs, we used unit costs collected from comparison districts in 2016 coupled with data on health services utilization from 2012 in SMGL-supported districts to estimate the costs before the start of SMGL. We collected data from health facilities, ministerial health offices, and implementing partners for the year 2016 in 2 SMGL-supported districts in each country and in 3 comparison non-SMGL districts . Incremental costs for maternal and newborn health care per SMGL-supported district in 2016 was estimated to be US$845,000 in Uganda and $760,000 in Zambia. The incremental cost per delivery was estimated to be $38 in Uganda and $95 in Zambia. For the districts included in this study, SMGL maternal and newborn health activities were associated with approximately 164 deaths averted in Uganda and 121 deaths averted in Zambia in 2016 compared to 2012. In Uganda, the cost per death averted was $10,311, or $177 per life-year gained. In Zambia, the cost per death averted was $12,514, or $206 per life-year gained. The SMGL approach can be very cost-effective, with the cost per life-year gained as a percentage of the gross domestic product (GDP) being 25.6% and 16.4% in Uganda and Zambia, respectively. In terms of affordability, the SMGL approach could be paid for by increasing health spending from 7.3% to 7.5% of GDP in Uganda and from 5.4% to 5.8% in Zambia. Secondarily, we assessed the sources of financing for the SMGL interventions. Findings from our analyses can inform stakeholder investments on cost-effective means to reduce maternal and perinatal mortality.,Uganda and Zambia have very high maternal mortality levels, despite the occurrence of substantial reductions, including downward national trends before and during the period of SMGL implementation.Understanding the maternal health ecosystem in a given geographic area through formal assessment of both public and private sectors.Using scarce resources rationally to address gaps and improve access to and quality of care.Addressing all 3 delays to care to ensure access to lifesaving care within 2 hours of the onset of a complication, focusing on the period of labor, delivery, and the 72 hours postpartum when women and newborns are most vulnerable.Decreasing missed opportunities by integrating MNH and HIV services.Counting, analyzing, and reporting all maternal and newborn deaths, and using findings to improve care.Against this background, SMGL was implemented in 2012 in an effort to dramatically and rapidly reduce maternal mortality in selected districts of Uganda and Zambia . The SMGL approach is based on context-specific solutions to maternal and, later, newborn health (MNH) problems. These solutions are identified and implemented through a coalition of partners, including governments, nongovernmental organizations, and the private sector.The SMGL approach is based on context-specific solutions to maternal and newborn health problems.With substantial subnational variation in maternal mortality ratios within these countries, SMGL targeted districts that had among the poorest maternal health indicators in each country.In the first year of implementation, SMGL was associated with a 35% reduction in the institutional maternal mortality ratio across the 2 countries.,We calculated the costs per maternal death averted and life-year gained by combining data on intervention costs that we compiled with direct outcome evaluation data from studies that previously documented maternal and newborn mortality associated with the SMGL approach. Health impact data in the SMGL-supported districts were collected in a separate evaluation of SMGL.The estimated costs of MNH interventions were assessed in a subset of the districts where the SMGL approach was implemented and compared to estimated costs in 2012, prior to SMGL interventions. Since costs were not directly collected in the SMGL-supported districts prior to SMGL implementation in 2012, we derived comparison costs from the 2016 unit costs  in neighboring districts where MNH programs were chiefly supported by country government efforts alone, to be consistent with the time frame for the effectiveness evaluation (end year as 2016). The estimated costs of MNH interventions were assessed in selected districts where the SMGL approach was implemented and compared to estimated costs in 2012, prior to SMGL interventions.Because cost data prior to SMGL implementation were not available, we assumed that SMGL would affect the unit cost of health services through both the scale (quantity utilized) and quality of services.Planned data collection included 2 of the 4 learning districts in each country. In Zambia, we randomly selected Mansa and Nyimba from the 4 SMGL-supported districts for inclusion in these analyses. Mansa became 2 separate districts (Mansa and Chembe) in 2012, and data were collected from both. We also selected 2 districts, Kapiri Mposhi and Mbala, for comparative purposes for the costing analyses. The intent was to conduct the costing study in districts used in an external evaluation of SMGL at the end of Phase 1, where 2 comparison districts were selected to be similar to SMGL-supported districts across a number of factors  that would also likely influence costs.In Uganda, we purposively included the greater Kibaale district  in the study because it was the only SMGL district to receive extensive support from one of the 2 main implementing partners. From the other 3 SMGL-supported districts, we selected Kyenjojo as the remaining rural district with a district hospital. Both districts received similar SMGL-supported interventions, with the exception of transport vouchers, which were implemented in Kyenjojo only.In each of the selected districts of Uganda and Zambia, we collected data from the district health office, the government hospital in that district, and 2 randomly selected government health centers. Overall, we collected data from 5 districts, 4 hospitals, and 6 health centers in Uganda , and 5 districts, 4 hospitals, and 8 health centers in Zambia. Data relating to MNH care activities in these districts were collected from district and provincial health offices (where appropriate) at the national level and from implementing partners, including 3 implementing partners in Uganda and over 30 in Zambia involved in MNH care, in the districts included in this analysis.Overall, we collected data from 5 districts, 4 hospitals, and 6 health centers in Uganda, and 5 districts, 4 hospitals, and 8 health centers in Zambia.The data collection specific to this study was exempted from the need for ethical approval by Abt Associates Institutional Review Board and from the University of Zambia Biomedical Research Ethics Committee in July 2017 because it did not include research on human subjects. The study received approval from the Makerere University of Public Health Higher Degrees, Research, and Ethics Committee in January 2018 and the Uganda National Council for Science and Technology  in February 2018.Data collection at health facilities occurred in July–August 2017 in Zambia and February–March 2018 in Uganda. Trained data collection teams extracted information on health facility area (square meters), staffing, service utilization, vehicles, and consumption of commodities from these facilities. Data were entered into Microsoft Excel templates designed for the study and were reviewed daily by data collection supervisors and again by the research team. Questions were sent to data collectors to verify information, and facilities were contacted again to clarify ambiguous information as needed. Similarly, structured templates were used to capture data at district health offices and, where appropriate, provincial health offices related to overall district health statistics  and activities related to MNH  during 2012–2016 for annualized start-up costs.,Data collection templates were constructed based on past analyses of expenditures in SMGL areasData were collected for all activities supporting MNH, whether or not they were “officially” part of SMGL. However, some activities were not assessed as part of the SMGL evaluation, including HIV/AIDS care or prevention for pregnant women and postpartum family planning outside the MNH clinics of health facilities, unless the SMGL program specifically included them.Data were collected for all activities supporting MNH, whether or not they were “officially” part of SMGL.,We used a 1-year analytic horizon for deaths averted  and a lifetime analytic horizon for life-years gained. We included costs from the health system perspective—that is, costs incurred by the health system and implementing partners. Costs incurred by patients and volunteers' time were not captured or included in the analysis. Capital items and start-up costs were converted into annual equivalent costs using standard formulas; we assumed a 3% discount rateWe used a financial approach to estimate the costs of activities presented in We employed a mix of top-down and bottom-up costing methods to estimate health facility costs.Total costs for MNH services for entire districts were estimated using the average unit costs from sampled facilities for different types of services  and multiplying the results by the total utilization of these services in a district. These figures include costs for inpatient admissions. For estimates of costs before the start of SMGL, we used utilization numbers from the SMGL-supported districts in 2012 and unit costs from comparison districts, while for 2016 we used utilization numbers and unit costs from SMGL-supported districts in 2016. We disaggregated these calculations by type of health facility. We then added costs incurred at the community level and “above service delivery costs”  to the facility-based costs. For SMGL districts, the programmatic costs and facility costs to address the first, second, and third delay total costs were added to derive the total costs for the districts. To convert costs incurred outside health facilities in comparison districts to costs incurred in 2012, we divided these costs by the number of facility deliveries in the comparison districts and then multiplied the results by the number of facility deliveries estimated to have occurred without SMGL in the SMGL districts. This calculation assumes that costs outside facilities varied directly with the number of deliveries at facilities; however, costs outside facilities in the comparison districts were a small proportion of all costs. For the 2012 cost estimates, we included observed community-level and above service delivery level costs from the comparison districts, under the assumption that these activities also likely existed in SMGL-supported districts before the start of SMGL. We calculated costs per facility delivery in 2012  and in 2016 . Incremental costs were calculated by taking the difference between the estimated total costs in 2016 and those in 2012. Sources of financing  were tracked throughout this exercise.Supplement 1).,,,To estimate the health impact, we used the facility-based maternal mortality ratios and perinatal death rates in 2016 in SMGL-supported districts multiplied by the reported number of facility-based deliveries for the cohort of women giving birth in 2016 to determine the number of deaths in 2016.,The number of deaths averted was estimated by subtracting the number of deaths in 2012 from the number of deaths in 2016. We estimated the incremental cost-effectiveness by dividing the incremental costs by the number of deaths averted in SMGL areas. Additionally, we estimated the potential remaining years of life for pregnant women and newborns at the time of death and calculated the cost per life-year gained using national life-expectancy data.We used locally published discount rates to calculate annual equivalent costs.We re-estimated the increased number of deliveries at health facilities using data from all 4 SMGL-supported districts rather than the 2 districts included in the costing. Chance variation in the increase in the number of deliveries at health facilities between districts may change the results.We re-estimated the proportion of facility deliveries by cesarean delivery, for the same reason as above and using the same method.We re-estimated incremental costs by considering all donor-supported costs as incremental costs (as opposed to using estimated 2012 costs). Although donor funds may displace some other sources of funding, this provides an upper-end estimation of the incremental costs in the absence of other district data.We re-estimated the cost of preventing a year of lost life considering a discount rate of 3% for future life-years, as suggested by the WHO-CHOICE guidance.We conducted sensitivity analyses to explore the potential impact of our assumptions on the results. Sensitivity analyses were done by changing input variable amounts and assessing how results were altered. The following scenarios and variables were considered for sensitivity analyses:We conducted sensitivity analyses to explore the potential impact of our assumptions on the results.Applying each of these 5 scenarios, we also calculated a “combined-case” scenario in which all the above scenarios were included at the least favorable value. Finally, we re-ran the analyses using mortality rates/ratios specific to the 2 SMGL-supported districts included in the costing.Average unit costs of a vaginal delivery in facilities in SMGL districts were lower or comparable to costs in facilities in non-SMGL districts in Uganda in 2016 . The oppSimilarly, cesarean delivery unit costs in Uganda were lower in health centers ($202) in SMGL districts than in health centers in the comparison district ($337). However, the costs were higher in Uganda hospitals ($163) in SMGL districts than in hospitals ($140) in the comparison district. At the referral hospital in an SMGL district, the cost of a cesarean delivery was $79 because the operating theater had a relatively high volume of services. In Zambia, the average cesarean delivery unit costs were lower in hospitals  in SMGL districts compared to hospitals ($508) in comparison districts.Average unit costs of an antenatal care visit were lower in health centers ($3.66 for level III and $3.59 for level IV) in SMGL districts than in health centers ($5.49 for level III and $5.07 for level IV) in the comparison district in Uganda. In contrast, hospitals in SMGL districts had higher average unit costs ($5.03) compared to hospitals ($4.60) in the comparison district for an antenatal visit. The cost structure in Zambia was different; health centers in SMGL districts had higher average unit costs of an antenatal care visit ($4.50) than facilities ($3.96) in comparison districts, while there were mixed results from the comparison of SMGL hospitals and non-SMGL hospitals.Each improved facility delivery cost about $104 in Uganda and $196 in Zambia.In 2012 in Uganda, total costs for MNH care were estimated to be about $650,000 per district, or almost $66 per facility delivery, while total costs for MNH care in Zambia in 2012 were estimated to be just under $425,000 per district or about $101 per facility delivery. Total costs for MNH care per district for the year 2016 were approximately $1.5 million in SMGL-supported districts in Uganda and almost $1.2 million in Zambia . This trIn 2016, donors covered about 49% of the MNH costs in Uganda and 45% of costs in Zambia in the SMGL-supported districts. In comparison districts, donors covered 2% (Uganda) and 3% (Zambia) of all costs in 2016. Costs incurred at private facilities accounted for 16% of costs in SMGL-supported districts in Uganda . Donors supported the majority of costs associated with the first and second delays and just under 30% of costs related to the third delay in SMGLsupported districts.Donors supported the majority of costs associated with the first and second delays and just under 30% of costs related to the third delay in both countries in SMGL-supported districts.In Uganda, the cost per facility delivery in 2016 in the SMGL-supported index districts was $38 higher than in 2012. Over 35% of the incremental cost went to support activities addressing the first delay, about 44% was spent on issues related to the third delay, 2% was spent on issues related to the second delay, and the remainder was spent on above community/facility costs for program support.Similarly, the cost per facility delivery in Zambia in SMGL-supported districts was about $95 more in 2016 than in 2012. Addressing the first delay accounted for about 14% of the incremental cost, and above community/facility costs for program support were associated with approximately 21% of the incremental cost per facility delivery. About 52% of the incremental cost in SMGL-supported districts in Zambia addressed the third delay.In Uganda SMGL areas, the institutional maternal mortality ratio was 534 deaths per 100,000 live births in 2012 and 300 in 2016. The institutional perinatal morality rate was 39.3 per 1,000 births in 2012 and 34.4 in 2016 in SMGL areas. The percentage of deliveries in facilities changed from 45.5% in 2012 to 66.8% in 2016, and the population cesarean delivery rate increased from 5.3% to 9.0%.In Zambia SMGL areas, the institutional maternal mortality ratio declined from 370 deaths to 231 deaths per 100,000 live births and the institutional perinatal morality rate declined from 37.9 to 28.2 deaths per 1,000 births from 2012 to 2016. The percentage of deliveries in facilities increased from 62.6% to 90.2% and the population cesarean delivery rate increased from 2.7% to 4.8%.Based on the number of facility deliveries in the 4 districts included in this analysis, scale-up of MNH activities is associated with averting 164 deaths in Uganda and 121 deaths in Zambia in the 2 SMGL-supported districts in 2016 included in this analysis . This trIn Uganda and Zambia, results related to the cost per death averted were otherwise most sensitive to assumptions about using all donor costs as incremental costs. For any given scenario, the cost per death averted remained less than $12,000 in Uganda and $14,300 in Zambia. In a scenario combining the 5 main sensitivity analyses, where all assumptions were moved to the least favorable cost-effectiveness scenario, the cost per death averted was around $12,411 in Uganda and $15,708 in Zambia. The cost per life-year saved was most sensitive to the assumption about whether to discount future life-years. When future life-years were discounted, the cost per life-year gained increased to $384 in Uganda and $460 in Zambia. In the scenario combining the 5 main sensitivity analyses, the cost per life-year gained was $462 in Uganda and $578 in Zambia.In the 4 SMGL-supported districts included in these analyses, scale-up of MNH interventions prevented an estimated 285 institutional maternal and perinatal deaths in 2016, or about 71 death per district per year (0.6 death averted per 100 facility deliveries in Uganda and 1.0 death averted per 100 facility deliveries in Zambia). GDP per capita is a commonly used benchmark to determine whether or not an intervention is highly cost-effective, with the cost per DALY averted below the GDP per capita used as the benchmark for being highly cost-effective.In the 4 SMGL-supported districts included in these analyses, scale-up of MNH interventions prevented an estimated 285 institutional maternal and perinatal deaths in 2016.Nevertheless, we found that the incremental cost per life-year gained in Uganda was $177, or 25.6% of the GDP per capita of $692, and the incremental cost per life-year gained in Zambia was $206, which is 16.4% of the GDP per capita of $1,257.We found that the incremental cost per life-year gained was $177 in Uganda and $206 in Zambia.A previous study assessing SMGL activities in Uganda suggested an incremental cost ranging from $28 to $104 per improved delivery, depending upon which activities were included in the costs, compared with our finding of about $38 per facility delivery.While recent estimates of unit costs of MNH activities are not available in Zambia, the unit costs found in this study are on the higher end of unit costs from other studies in Uganda. For example, a recent review found the cost of antenatal care in Uganda was about $5.90 at health centers and $6.40 at hospitals per woman,We did not see a marked change across the board in unit costs of services between SMGL-supported districts and comparison districts when we included only costs incurred at health facilities. In many cases, unit costs were lower in SMGL-supported districts. This was likely because of higher patient volumes in SMGL-supported facilities, with the increased efficiency in the use of capital and overhead costs offsetting the costs of increasing the quality of services. The exception was for vaginal delivery in Zambia, where unit costs were mostly higher in SMGL-supported districts than in comparison districts, but also where there was less difference in the number of deliveries between 2012 and 2016 than in Uganda. When we included costs incurred outside health facilities, including training, mentoring, and community mobilization—that is, the cost of an improved facility delivery—the cost per facility delivery in SMGL-supported districts was substantively higher than in comparison districts.Funding for reducing the first delay constituted 36% of incremental costs in Uganda and 14% of incremental costs in Zambia, representing 20% and 10% of total costs in SMGL-supported districts, respectively. In comparison districts, the cost of activities addressing the first delay was either nonexistent (Uganda) or marginal ($1.81 per facility delivery in Zambia). Costs for the activities addressing the second delay were 4% and 9% of total costs in SMGL-supported districts in Uganda and Zambia, respectively. While funding for the second delay was similar in the SMGL-supported and comparison districts in Uganda , it was substantially higher in Zambia, where costs for maternity homes were a main cost driver for the second delay. For each facility delivery, $17.73 was spent on activities addressing the second delay in Zambia SMGL-supported districts, contrasting with $2.44 in comparison districts. The increase in costs per facility delivery was less marked (in percentage terms) for activities addressing the third delay, possibly representing either efficiencies, as noted above, or displacement of other funds. In terms of the total incremental costs, the third delay used the highest amount of resources in Uganda  and in Zambia . However, the results suggest that spending about 20% to 25% of MNH budgets to address the first 2 delays—critical delays that can prevent women from accessing care in a timely way—can be enough to improve receipt of timely facility care at birth. While securing and ensuring funding for activities to address the first 2 delays is critical, the results also suggest that in Uganda and Zambia, funding for facility deliveries was inadequate in 2012 to provide sufficient quantity and quality of care, with donors supporting more than 25% of costs addressing the third delay in SMGL districts in both Uganda and Zambia in 2016.The results suggest that spending about 20% to 25% of MNH budgets to address the first 2 delays can be enough to improve receipt of timely facility care at birth.This study is limited by use of comparison districts that were assessed only at the end of the SMGL program. These districts serve as an imperfect proxy estimate of the cost of MNH services before the start of the SMGL program. In addition, use of before and after data to estimate the effects of the scale-up of MNH services is subject to confounding due to secular trends. Although we tried to account for secular trends using national data, the national trends may not have been realized in the SMGL districts over the same time period.Because data from 2016 in comparison areas were used as proxies for unit costs in SMGL-supported districts in 2012, we assessed data from 2016 in comparison districts with data from SMGL-supported districts in 2012 to ensure comparability. In Uganda, there were about 700 births per facility in SMGL-supported districts in 2012 and 500 births per facility in comparison districts in 2016, while in Zambia, there were about 225 births per facility in both 2012 in SMGL-supported districts and 2016 in comparison districts. In Uganda, 6% of facility births were by cesarean delivery in the SMGL-supported districts in 2012, compared with 9% in comparison districts in 2016, while in Zambia the percentage of facility births by cesarean delivery was 7% and 3%, respectively. Because data from comparison districts suggest a close match with intervention districts before the start of SMGL in some cases but a notable difference in other instances, we addressed potential biases in sensitivity analyses by using available data on likely ranges for changes in facility-based deliveries, cesarean deliveries, and incremental costs per facility delivery. In all cases, conclusions did not change substantively.However, the effects presented here could potentially be underestimates for several reasons. Assessing progress in reducing facility maternal and perinatal mortality during the initiative required using facility data and data abstraction protocols. In 2012, each country faced the immediate challenge of how to produce baseline measurements of maternal and perinatal mortality in the period immediately before the initiative began and comparable measurements during the initiative, when data quality improvements were institutionalized. At baseline, each country used its existing data systems and infrastructure to devise its own independent data-collection approach. Although the definitions of indicators were standard, the quality of primary data used to calculate the number of maternal and perinatal deaths was substantially lower at baseline than at endline in both countries. In addition, differences in data collection existed between Uganda and Zambia. Thus, some deaths were likely missed in the baseline count, which would bias our results downward. Further, the proportion of deliveries in facilities increased over time, but we applied the facility-based death ratios/rates to all births. To the extent that women who would have given birth at home without SMGL would have worse outcomes than were observed for facility births, we underestimate the effects of the program.,Lacking data, we have not tried to incorporate these effects into the analyses. Further, the complete effects of the program, which may include increasing staff morale and their ability to deliver other interventions (such as family planning or prevention/elimination of mother-to-child transmission of HIV), were not captured in the effect estimates. While we did not assess changes in patient payments to access services, we also did not include the potential cost savings  resulting from preventing a maternal or newborn death.,,Donors spent upwards of $733,000 per district in Uganda and $538,000 per district in Zambia in total annual equivalent costs, and in the first year of SMGL $2 million and $1.5 per district in real budgetary expenditures. These findings are in keeping with a previous study assessing SMGL expenditures .While the results from Uganda and Zambia were similar in terms of their cost-effectiveness, the sensitivity analyses looking at results only for districts with cost data indicate that heterogeneity would certainly exist in applying the results to other settings and within countries themselves. SMGL was targeted to areas within Uganda and Zambia with high maternal mortality, with some activities tailored to each district. Similar targeted approaches are likely necessary in other settings, which may affect the cost-effectiveness in any particular setting. Further, the costs presented here do not account for potential changes to costs structures, demand for services, and average unit costs over time. In the future, increased uptake of family planning, further increases in demand for and use of services, and so forth will likely change the unit costs of delivering MNH services as well as the mix of activities needed. Thus, the cost-effectiveness of district health strengthening approaches such as SMGL will likely also change over time.The SMGL project could be paid for by increasing health spending from the 7.3% of GDP in 2015 (in Uganda) and 5.4% (in Zambia) to 7.5% and 5.8% of GDP, respectively.This study adds to the literature by presenting actual costs and effects of a health systems strengthening approach that addressed the 3 key barriers to receiving MNH care. We find that the approach costs about $177 to $206 per year of life gained, depending on the context. Ministries of Health and donor agencies have already demonstrated a willingness to pay this amount per year of life gained; for example, first-line antiretroviral therapy cost over $200 per person per year across 5 countries (including Zambia) in sub-Saharan Africa in 2010.


Phlebotomus neglectus males. The virus was provisionally named the Drin virus after a river near the locality of Kukës, where the infected sand flies were trapped. Genetic and phylogenetic analysis revealed that the Drin virus is closely related to the Corfou (CFUV) virus, isolated in the 1980s from Phlebotomus major sand flies on the eponymous island of Greece, and may also be involved in human infections because of its similarity to the sand fly fever Sicilian virus. The latter justifies further studies to specifically address this concern. Together with recent findings, this study confirms that Albania and the Balkan peninsula are hot spots for phleboviruses.Phlebotomine sand flies are generalist vectors with significant implications for public health. They are able to transmit phleboviruses that cause sand fly fever, headaches, or meningitis in humans. Albania is a country in Southeast Europe with a typical Mediterranean climate which provides convenient conditions for the presence of sand flies. Hence, the circulation of phleboviruses, such as the Toscana and Balkan viruses, has been recently described in the country. We followed a virus discovery approach on sand fly samples collected in 2015 and 2016 in seven regions of Albania, with the aim to investigate and characterize potentially circulating phleboviruses in phlebotomine sand flies. A presumed novel phlebovirus was detected in a pool consisting of 24  Phlebovirus, family Phenuiviridae, order Bunyavirales) can be classified into three serological complexes, which are also regarded as taxonomic species or tentative species—the Sandfly fever Naples phlebovirus species (SFNV), the Salehabad phlebovirus species, and the Sandfly fever Sicilian phlebovirus tentative species (SFSV). The SFNV includes the following viruses: Arrábida (ARRV), Balkan (BALKV), Fermo (FERV), Gordil (GORV), Granada (GRAV), Massilia (MASV), Punique (PUNV), Saddaguia (SADV), Saint-Floris (SAFV), sand fly fever Naples (SFNV), Tehran (THEV), Toscana (TOSV), and Zerdali (ZERV) . The Salehabad phlebovirus species includes Adana (ADAV), Adria (ADRV), Alcube (ALCV), Arbia (ARBV), Arumowot (AMTV), Medjerda Valley (MVV), Odrenisrou (ODRV), Olbia (OLBV), Salehabad (SALV), Bregalaka (BREV), and Zaba (ZABAV) . The SFSV tentative phlebovirus species is a serocomplex which includes several phlebovirus species that are yet to be recognized by the International Committee on Taxonomy of Viruses (ICTV). The SFSV and Corfou (CFUV) phlebovirus tentative species are antigenically and genetically close to each other and may be grouped together as a unique species [According to their antigenic relationships, old world sand fly-borne phleboviruses , followed by P. perfiliewi (14.4%), P. papatasi (4.6%), P. tobbi (3.6%), P. similis (1.8%), P. simici (<1%), Sergentomyia dentata (<1%), and Sergentomyia minuta (<1%) [Albania has a typical Mediterranean climate that provides convenient environmental conditions for the active circulation of sand flies between May and November. To date, a total of eight sand fly species have been described in Albania, ta (<1%) ,12.Salehabad phlebovirus species [Recently, several phleboviruses were identified in specific areas of Albania—the BALKV virus, belonging to the SFNV species and the ADRV virus, belonging to the  species ,13.Our aim was to investigate and characterize the phleboviruses in phlebotomine sand flies collected in more than one region in Albania from June to September of 2015 and 2016.2 baited traps  were used to collect sand flies in seven regions of Albania from June to September of 2015 and 2016 ) with 3 mm tungsten beads added into tubes. Sand fly tissues were homogenized by a Mixer Mill MM300 , and the mixture was centrifuged at 5800× g for 10 min.A volume of 600 μL of Eagle’s minimal essential medium (EMEM) , 1% penicillin–streptomycin, 1% (200 mM) 2 atmosphere. The nucleons were examined daily for the presence of a cytopathic effect (CPE) and passaged 8 times. In each passage, 200 μL of supernatant medium was collected from the nucleons and tested by reverse transcription PCR (RT-PCR) with stratifin (SFN), Nphlebo, and Lambert primers [A volume of 50 μL homogenate supernatant was inoculated into 450 μL Vero cells in enriched EMEM without FBS. The cells were then incubated at room temperature for 1 h. After 1 h, 2.5 mL of fresh EMEM with 5% FBS was added and the nucleons incubated at 37 °C in a 5% CO primers ,15,16,17A volume of 2 μL of the original homogenate supernatant was used in an attempt to infect 2 to 3 day-old Oncins France 1 (OF1) mice intracerebrally. The mice were observed for signs of disease and sacrificed on days 6 to 14 of the experiment. These animal experiments were carried out according to the animal experiment regulations of Aix-Marseille Université under the license A1301309. The RNA was extracted from the brain tissues of the mice by using a QIAcube HT system with a QIAamp 96 Virus QIAcube HT Kit  according to the manufacturer’s instructions and studied for the presence of the Drin virus.A volume of 200 μL homogenate supernatant was used for nucleic acid extraction. For the 2015 sand fly samples, the extraction was performed by a BioRobot EZ1-XL Advanced  with a Virus Extraction Mini Kit (Qiagen). The samples from 2016 were extracted using QIAcube HT system with a QIAamp 96 Virus QIAcube HT Kit .RT-PCR was performed with the Super Script III (Thermo Fisher Scientific) kit using three different systems: (1) the Nphlebo1S/1R system targeting the polymerase gene in the L RNA segment, (2) the SFNV1S/1R system targeting the nucleoprotein gene in the S RNA segment, and (3) the Lambert Phlebovirus system, also targeting the S RNA segment ,15,16,17Nested PCRs were performed with a DreamTaq kit  with Nphlebo2 and SFNV2 primers. The following cycle was used for the Nested-PCRs: 94 °C for 5 min, followed by 40 cycles at 94 °C for 30 s, the annealing temperature for 45 s, and 72 °C for 30 s, with a final elongation step at 72 °C for 7 min.Real-time quantitative RT-PCR (RT–qPCR) was performed with the EXPRESS qPCR SuperMix Universal  kit and the Quant Studio 12K Flex System. The cycling program of the RT-qPCR reaction consisted of 50 °C for 5 min and 94 °C for 20 min, followed by 40 cycles at 95 °C for 3 s, then 60 °C for 30 s. Specific primers were used to screen for the SFSV group viruses and TOSV ,18.The Drin virus was identified as closely related to the Corfou virus based on the partial sequence obtained from the Nphlebo primers in the polymerase (L RNA).Because of the closeness between the polymerase partial sequences of the Drin and CFUV viruses, we designed new primers using the CFUV virus sequences as a reference. We assumed that these primers would also amplify the Drin virus segments .Additionally, we designed real-time RT-qPCR assays specifically targeting the Drin virus L-, M- and S-segments to test all the pools to check whether the Drin virus sequences were present in any of the other analyzed pools . The samThe positive PCR products were purified using a QIAquick PCR purification kit  and sequenced with next-generation sequencing (NGS) using an AU4c Ion-Torrent PGM platform as described .The sequence data was analyzed through CLC Genomics Workbench version 11 software . Homologous sequences from phleboviruses were aligned with homologous sequences of interest using Clustal W in Molecular Evolutionary Genetics Analysis 6 (MEGA6) software . The evoc oxidase I (COI) and cytochrome b (Cytb) genes, respectively, as previously described [To identify the sand fly species constitutive of the Drin virus positive pool, barcoding PCR was performed with 3 μL of nucleic acid to amplify partial regions of the cytochrome escribed ,22. The COI gene and 397 reads for the Cytb gene. All 7307 reads 100% matched the P. neglectus sequences, and thus demonstrated that the 24 sand flies contained in Pool #17 belonged to the P. neglectus species.A total of 2502 sand flies were tested for phlebovirus presence, 533 collected in 2015 , and 1969 in 2016  in seven different regions of Albania . They weVero cells inoculated with supernatant corresponding to Pool #17 were examined daily for CPE. Eight blind passages were done, but no CPE was observed. In addition, each passage was tested for the presence of Drin virus RNA using the SFN, Nphlebo, and Lambert sets of primers; the results were consistently negative, indicating that the Drin virus was not isolated in cell culture. For this reason, we decided to try virus isolation by using intracerebral (IC) inoculation into suckling mice. A total of 25 baby mice were injected with sand fly homogenate supernatant with the attempt to isolate the Drin virus. None of the mice developed clinical signs, which suggested that the Drin virus was not replicating in their brain tissues. Baby mice were euthanized at 7 days post-IC injection, the brain was then collected postmortem, with nucleic acid extraction performed as described above and tested with different PCR assays. All results were negative, showing that the Drin virus had not been isolated.Partial sequences of the three RNA segments  were amplified and sequenced. In all cases, the Drin virus sequences were most closely related to, but clearly distinct from, those of the CFUV virus .Regardless of the gene used for analysis, the Drin virus is grouped with the CFUV and Toros viruses (with 99–100% bootstrap support), the three viruses forming a sister cluster to the group including the Dashli virus and strains of SFSV .All these viruses form a solid monophyletic cluster which is consistent with the proposal that they should all be placed within a single species, one that is yet to be recognized by the ICTV as a SFSV species.Phlebovirus. Recently, the isolation of the Dashli virus in Iran has been an opportunity to propose to group SFSV, CFUV, and Dashli viruses within a single species putatively named Sandfly fever Sicilian phlebovirus species [The viruses belonging to the SFSV and CFUV tentative phlebovirus species have previously been described in Italy, Turkey, Cyprus, Greece, Iran, and Ethiopia, but they have never been detected in Albania or the other countries of the Western region of the Balkans ,24,25,26 species .P. neglectus species which is the predominant species in that area. Other studies have demonstrated that phleboviruses were potentially transmitted by several distinct species of sand flies [COI and Cytb genes of sand flies is an efficient approach for species identification within a pool of insects. Here, all sand flies included in Pool #17 were P. neglectus, suggesting that this species is capable of transmitting the Drin virus between sand flies and possible vertebrate hosts. As P. neglectus is widespread in the Balkan peninsula, in Eastern Europe, and in Turkey [In this study we identified a novel phlebovirus, tentatively named the Drin virus after the eponymous river in the Kukës region of Albania. The virus was identified in a pool containing only male sand flies belonging to the nd flies . The secnd flies ,28,29,30nd flies . Assuminnd flies . There ind flies . Sand flnd flies , NGS anan Turkey , and becn Turkey  and Toscn Turkey , competeGene by gene comparative distance analysis showed that pairwise distances of the Drin virus versus CFUV and Toros viruses were consistently lower than the lowest distances observed between Drin virus and SFSV–Dashli virus. Detailed genetic distance analyses have been done for the SFSV and CFUV phlebovirus tentative species by Alkan et al. .Larroussius). Evidence pointing to the Drin and CFUV viruses as two distinct viruses—they have been discovered in different species of sand flies, they were discovered in different countries, and the Drin virus was found in sand flies trapped in the northern part of Albania which has no contact with Corfou island that faces the Ionian coasts of Albania. It is not known whether cross-neutralization exists for the Drin and CFUV viruses, and the isolation of the Drin virus is required to conduct the proper experiments to address this question.The limited genetic divergence between partial sequences of the Drin and CFUV viruses questions whether they are two distinct viruses or two strains of the same virus. Since taxonomy does not regulate delineation below the species level, there may be contradictory elements. In favor of considering that the Drin and CFUV viruses are two strains of the same virus—they are genetically very close, and they have been detected in two species of sand flies belonging to the same subgenus  showing that SFSV was circulating in Portugal, but there is not any seroprevalence survey to document this in Albania .Sandfly fever Naples phlebovirus species, Adria, Zaba and Bregalaka viruses for the Salehabad phlebovirus species, CFUV and Drin viruses for the tentative SFSV phlebovirus species.As a consequence, there are representatives of the three main species of sand fly-borne phleboviruses in Albania  and in the Balkan as well: Toscana and Balkan viruses for the SFSV has been described in an extended geographic area from southwestern Europe to the Middle East including northern Africa. SFSV epidemics were recorded in Italy, Cyprus, Turkey, and Ethiopia ,27. Furt


Repetitive passive movement (PM) affects corticospinal excitability; however, it is unknown whether a duty cycle which repeats movement and rest, or subjects’ conscious attention to movements, affects corticospinal excitability. We aimed to clarify the effect of the presence or absence of a duty cycle and subjects’ attention on corticospinal excitability. Three experiments were conducted. In Experiment 1, PM of the right index finger was performed for 10 min. Three conditions were used: (1) continuous PM (cPM) at a rate of 40°/s; (2) intermittent PM (iPM) with a duty cycle at 40°/s; and (3) iPM at 100°/s. In conditions 1 and 3, motor evoked potential (MEP) amplitude was significantly reduced. In Experiment 2, PM was performed for 30 min: condition 1 comprised cPM at a rate of 40°/s and Condition 2 comprised iPM at 40°/s. MEP amplitude significantly decreased in both conditions. In Experiment 3, PM was performed for 10 min: condition 1 comprised paying attention to the moving finger during iPM and Condition 2 was similar to Condition 1 but while counting images on a monitor without looking at the movement finger, and Condition 3 comprised counting images on a monitor without performing PM. MEP amplitude significantly increased only under Condition 1. Thus, afferent input from movements above a certain threshold may affect corticospinal excitability reduction. Furthermore, corticospinal excitability increases when paying attention to passive finger movement. Corticospinal excitability decreases after passive movement  intervention significantly increases corticospinal excitability  participated in this study. Experiment 1 utilized 15 healthy adults ; Experiment 2 utilized 10 healthy adults ; and Experiment 3 utilized 14 healthy adults . No subjects had any central neurological or psychological disorders. This study was approved by the Ethics Committee of Niigata University of Health and Welfare and was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent before participating in this research.The target muscle was the right first dorsal interosseous muscle (FDI), which was monitored with disposable Ag/AgCl electrodes in a belly−tendon montage. The earth electrode was wrapped around the right forearm. Electromyography (EMG) data were recorded using a surface electrode  connected to an amplifier . The amplified EMG signal was digitized using an A/D converter . The sampling frequency was 4 kHz, and filtering was also performed using a 20 Hz high-pass filter. Data was imported into a personal computer and stored using analysis software .Motor evoked potential (MEP) was measured by TMS as a means of evaluating corticospinal excitability. A Magstim 200  and a figure 8 coil  were used for MEP measurement. The coil was placed tangentially to the scalp, and the handle part of the coil was placed tangentially at approximately 45° behind the midline. The magnetic stimulation site was the finger area on the left M1 and was defined as the site (hot spot) where the MEP was most induced by the right FDI. Magnetic resonance imaging and Visor 2 TMS Neuronavigation  were also used for identification of the hot spot on the right FDI. The position, direction and angle of the coil were made constant before and after the intervention. The magnetic stimulation intensity was defined as the intensity at which MEP amplitude was induced to about 1 mV at rest. MEP was measured 15 times before intervention (Pre) as well as immediately and 5 and 10 min after intervention . The magnetic stimulation interval was set to 5–6 s.A custom-made PM control device  that can control motion speed and angle was used in all three experiments.Three PM conditions were used . The movThe PM condition were used and based on Condition 1 and Condition 2 in Experiment 1; however, both movement times were increased to 30 min . TherefoFocusing on the presence or absence of the subject’s attention, three conditions were utilized . ConditiIn Condition 2 (no attention), PM was the same as in Condition 1 but subjects were asked to count the number of circles displayed on a monitor placed directly in front of their faces.The image sequence displayed on the monitor is shown in In the Condition 3 (control), no PM was performed, and similar to Condition 2, the number of circles randomly displayed on the monitor was counted.In Experiments 1, 2, and 3, 15 MEPs were measured for Pre, Post-0, Post-5, and Post-10 using TMS. All the experiments were performed in the afternoon, and each condition was randomly assigned on a different day. In all experiments, the subjects sat in a reclining chair to which a headrest was attached, with their right forearms on a table while maintaining a comfortable posture at all times.LabChart 7 software (AD Instruments) was used for MEP analysis. The maximum and minimum values of the 15 MEP waveforms obtained before and after the intervention for each condition were excluded, and the MEP amplitudes of the remaining 13 waveforms were averaged. The peak-to-peak value was then calculated as the MEP amplitude value.PASW statistical analysis software Ver. 21  was used for statistical analysis. Two-way repeated measure analysis of variance (ANOVA) was used to compare MEPs between INTERVENTION and TIME factors  in each experiment. Mauchly’s test of sphericity was used to analyze the sphericity of the data obtained in each experiment. When the Mauchly’s test of sphericity could not be assumed, the Greenhouse–Geisser correction statistic was used. When a main effect or interaction was observed, multiple comparisons were performed using the Bonferroni method. The level of significance was set at 5%.F = 5.019, P = 0.014, partial η2 = 0.264), of the main effect of the TIME factor  = 5.443, P = 0.010, partial η2 = 0.280) and of their interaction  = 3.277, P = 0.006, partial η2 = 0.190). Results of post hoc test showed that there was significant decrease in MEP in Post-0 and Post-5 compared with Pre in Condition 1 (P < 0.01) and 3 (P < 0.05), whereas there was no significant change in MEP in Condition 2.Changes in MEP over time are shown in F = 23.652, P < 0.001, partial η2 = 0.724), but no of the main effect of the INTERVENTION factor  = 1.729, P = 0.221, partial η2 = 0.161) or their interaction  = 0.632, P = 0.601, partial η2 = 0.066). Because the main effect of the TIME factor was significant, post hoc test was performed using the average results from the two conditions and found a significant decrease in MEP in Post-0, Post-5, and Post-10 compared with Pre (P < 0.01).Changes in MEP over time are shown in F = 5.079, P = 0.014, partial η2 = 0.281) and the interaction between INTERVENTION and TIME  = 2.858, P = 0.014, partial η2 = 0.180) but no significant difference in main effect of the TIME factor  = 1.678, P = 0.188, partial η2 = 0.114). Results of post hoc test showed that Condition 1 exhibited a significant increase in MEP amplitude at Post-10 compared with Pre (P < 0.01), but no significant MEP amplitude changes were observed in Conditions 2 and 3.Changes in MEP over time are shown in The number counted in all conditions was 240. In Conditions 1 and 3, only one person made a mistake in the count number once. In Condition 2, no subject made a mistake in the count number.This study investigated the effect of the presence or absence of a duty cycle, which provides movement and rest during repetitive PM, and the influence of paying attention to PM on corticospinal excitability. As a result, in Experiment 1, corticospinal excitability decreased over 600 continuous repetitive PMs and 600 intermittent PMs. However, corticospinal excitability did not change over 240 intermittent repetitive PMs. The cortical excitability temporarily declines after light repetitive voluntary movement (Zanette et al., Experiment 2 used two conditions from Experiment 1, those being continuous repetitive PM, where corticospinal excitability decreased, and intermittent repetitive PM, where corticospinal excitability did not change, and movement time was increased from 10 to 30 min. Therefore, continuous repetitive PM consisted of 1,800 movements overall, whereas intermittent repetitive PM consisted of 720 movements. As a result, corticospinal excitability decreased in both conditions, indicating that corticospinal excitability decreases with increasing numbers of movements, even with intermittent repetitive PMs where corticospinal excitability did not change with 240 movement repeats. Therefore, it seems that changes in corticospinal excitability do not depend on the presence or absence of the duty cycle but rather are influenced by the number of movements. It is reported that the cortical excitability temporarily decreases after repetitive PM (Miyaguchi et al., A, central cholinergic are involved in generation of SAI (Ziemann et al., The results from the first two experiments suggested that the presence or absence of a duty cycle does not affect changes in corticospinal excitability. Therefore, in Examination 3, we examined the influence of paying attention to PMs on corticospinal excitability. As a result, when attention was paid to the moving finger during PM, corticospinal excitability increased, whereas corticospinal excitability did not change under conditions where attention was not directed to passive finger movements. Macé et al.  reportedThis study demonstrated that continuous repetitive PMs and intermittent repetitive PMs consisting of a total number of 600 movements reduced corticospinal excitability but a total of 240 intermittent repetitive PMs did not. Furthermore, 1,800 continuous repetitive PMs and 720 intermittent repetitive passive exercises decreased corticospinal excitability. Therefore, corticospinal excitability was affected by afferent input based on the number of movements above a certain threshold. Furthermore, corticospinal excitability increased when subjects directed their attention to the moving finger during PM.Anonymized data for the manuscript are available on request. Please contact the corresponding authors for the same.HO and ST conceived the study and designed the experiments. ST, RS and MP performed the experiments and performed statistical analysis. SM and SK performed data interpretation. KS, YI and NO helped in writing the manuscript. HO and ST wrote the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tick-borne flaviviruses have a global distribution and cause significant human disease, including encephalitis and hemorrhagic fever, and often result in neurologic sequelae. There are two distinct properties that determine the neuropathogenesis of a virus. The ability to invade the central nervous system (CNS) is referred to as the neuroinvasiveness of the agent, while the ability to infect and damage cells within the CNS is referred to as its neurovirulence. Examination of laboratory variants, cDNA clones, natural isolates with varying pathogenicity, and virally encoded immune evasion strategies have contributed extensively to our understanding of these properties. Here we will review the major viral determinants of virulence that contribute to pathogenesis and influence both neuroinvasiveness and neurovirulence properties of tick-borne flaviviruses, focusing particularly on the envelope protein (E), nonstructural protein 5 (NS5), and the 3′ untranslated region (UTR). Flavivirus genus (family Flaviviridae), which includes the more well-known mosquito-borne flaviviruses (MBFV): West Nile virus (WNV), dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever (YFV), and Zika virus. The TBFVs generally circulate in Europe and Asia and are vectored by Ixodidae ticks, although alimentary transmission has also been documented . Th. Th80]. The 3′VR starts immediately downstream of the stop codon of the viral ORF and upstream of the conserved region and is extremely heterogeneous in both length and sequence in natural isolates ,91,92. T50 < 10), resulting in an undetectable increase in virulence following deletion of the VR. Initially, the VR was not thought to affect flavivirus biology, as the entire VR of the TBEV-Eu Neudoerfl strain was deleted without any significant effect on virus pathogenicity . Recent Secondary structure predictions showed three SL structures in the Oshima VR, designated SL3, SL4, and SL5 by Sakai and colleagues  and TIA-1 related protein (TIAR) were also found to bind the 3′UTR of Neudoerfl and exhibited a suppressive effect on viral replication . AlthougSakai and colleagues also demonstrated the role of a naturally introduced poly(A) tract as a virulence factor. A 35 nt stretch of A was introduced into the full-length 3′VR of Oshima-IC, where Sofjin-IC contains a deletion. The recombinant Oshima-IC-poly(A) mutant resulted in the same level of mortality as seen with Sofjin-IC infection . The rolIn addition to its role in genome cyclization and translation, the 3′UTR is important in the synthesis of subgenomic flavivirus RNA (sfRNA). Following decapping of +ssRNA or upstream cleavage in the genomic RNA, host 5′–3′ exoribonuclease (XRN1) generates sfRNA  via stalling on SL structures during viral RNA degredation ,100,101.Although TBFVs are emerging and reemerging pathogens with a significant disease burden, they receive much less attention than their MBFV counterparts. Determining virulence factors and uncovering the mechanisms behind TBFV pathogenesis will be critical for the development of vaccines and therapeutics. The ability to influence virulence, in particular neurovirulence, without interfering with immune activation is of particular importance in the development of safe and effective vaccines against TBFVs. To this end, residues affecting neurovirulence remain to be identified. As discussed previously, no mutations in the E protein have been identified that reduce neurovirulence specifically, without affecting peripheral infectivity and neuroinvasiveness as well. Identifying high affinity or cell specific entry receptors will also be of importance. In addition, the 3′VR and sfRNA production may play a major role in TBFV pathogenesis but remain to be explored in greater depth. Uncovering host–virus protein interactions responsible for immune activation and the development of neurological symptoms and damage will be critical for developing therapeutics, as treatment for TBFV infection is currently supportive.