The purpose of this study is to summarize recent advances in the use of broadly neutralizing antibodies (bNAbs) as therapeutics in human clinical trials and in non-human primate (NHP) models. We seek to highlight lessons from these studies with an emphasis on consequences to the virus and immune system.In the past 10 years, advances in HIV-1 trimer structure and B cell isolation methods have precipitated the identification of “new-generation” anti-HIV antibodies with broad and potent neutralization. In the past 2 years, the concept of using these bNAbs as therapeutic tools has moved from NHP models into human clinical trials. These trials have investigated the effects of bNAb infusions into patients chronically infected with HIV-1, while the NHP model has investigated treatment during acute infection.Through this work, the relationship between in vitro breadth and potency and in vivo clinical effect, although unresolved, is gradually being elucidated. These results emphasize the need for combination antibody therapy. Antibodies have been used as passive therapy for infectious diseases since the treatment of diphtheria with serum in the late 1800s . As for The feasibility of antibodies for treatment of HIV-1 depends on many factors including manufacturability, cost, and delivery, with current research investigating antibody development and optimization to exploit the full range of antibody-associated antiviral effects. Notably, all antibody functions  could likely contribute to adjunctive therapy with current anti-retroviral treatment (ART) regimens , 8. ViruTo date, the correlation between in vitro neutralization and in vivo clinical effect is not fully defined, and the effect of antibody treatment on the balance between the virus and immune system, with particular reference to viral escape and the effect on viral set point, remains unknown Fig. . Viral eTwo studies demonstrated the feasibility of passive immunization with bNAbs during chronic infection with simian-human immunodeficiency viruses (SHIVs) in rhesus macaques (RMs). Both tested bNAbs, singly and in combination, that targeted the HIV-1 envelope receptor CD4 binding site (CD4bs) (3BNC117 and b12) or the V3-glycan supersite (10-1074 and PGT121). Because 3BNC117, 10-1074, and PGT121 either have or will be used in human clinical trials, a comparison between human and NHP data will be possible.AD8 and after 12 weeks of infection, during virus set-point, they infused four RMs with 10 mg/kg intravenously of either 3BNC117 or 10-1074 [AD8 virus, with IC50 values ≤0.2 μg/ml. Infused RMs suppressed virus within 4–14 days and maintained suppression for 2–10 days. Single-genome amplification (SGA) of rebound virus on days 23 and 28 post-infusion revealed resistance mutations to 10-1074 (loss of N332 glycan), but no obvious changes within the 3BNC117 epitope. Antibodies were next co-administered to five RMs infected for at least 3 years. In the three RMs whose baseline VL was 1 × 104 copies/ml or lower, virus was suppressed by day 6 or 7 and remained undetectable until rebound between days 17 and 42. In the two RMs with virus >1 × 105 copies/ml, VL decreased until day 6 or 20 and then began to increase. SGA on rebound viruses on days 28, 49, or 57 revealed no obvious resistance mutations within antibody epitopes.The first study by Shingai et al. infected RMs with SHIV 10-1074 . These aSF162P3-infected RMs [SF162P3 in vitro, this virus was more resistant to 3BNC117. This observation correlated with viral suppression within 7 days and maintenance of suppression for 42–56 days in the four macaques infused with PGT121. Remarkably, in one of these PGT121-infused animals, viral control was observed for more than 200 days in the absence of antibody. The four macaques infused with 3BNC117, however, experienced a smaller decline in VL, without full suppression, and returned to set-point by day 20 post-infusion, reflecting the insensitivity of the virus to this bNAb. Combination therapy worked well, as demonstrated by the infusion of 10 RMs with a cocktail of anti-HIV bNAbs  and >2 log decrease in VL. The two RMs with the highest baseline VL (around 5 × 105 copies/ml) did not fully suppress virus replication and VL began to increase around day 15. The other eight RMs remained suppressed for multiple days and began to rebound between days 20 and 84 post-infusion. Together, in all RMs infused with PGT121, the time to virus rebound was correlated with baseline VL. Despite these differences in virus rebound SGA of virus from seven RMs at week 8 post-infusion revealed no obvious resistance mutations, although changes in and around the epitopes of these antibodies could be detected. In addition, the effect of the 3BNC117 in these cocktail antibody infusions was debated due to its reduced capacity to neutralize the circulating virus.Barouch et al. reported similar results in SHIVcted RMs . RMs werSF162P3 after infused mAb was cleared, as well as reduced activation of virus-specific T cell responses and a slightly lower median set-point VL (0.61 log lower) was observed in antibody-treated RMs.This study highlighted hints of indirect effects of antibody treatment on the immune system and the virus. A trend of increased neutralization potency by autologous antibodies against SHIVTogether, these NHP studies demonstrate that viral escape from infused antibody can occur, but also that it is possible for a single bNAb to suppress virus replication. In this case, virus rebounds to set-point as antibody levels wane, with little evidence of virus escape. Furthermore, low baseline VL, high viral neutralization sensitivity, and bNAb combination therapy were associated with longer suppression of virus replication. However, within this NHP model, the development of anti-drug responses led to faster clearance of human antibody concentrations in the RM serum. In addition, the chronic SHIV quasi-species in the NHP model may be less diverse than HIV-1 in humans; therefore, neutralization sensitivity of the circulating virus may be easier to define in RMs leading to a clearer relationship between in vitro and in vivo potency.10. Mean VL decrease was 1.48 log10 and median nadir was 7 days. The magnitude of the decline was found to be related to the baseline VL and pre-existing sensitivity of the virus to the antibody. Sensitivity was tested by bulk PHA stimulation of autologous PBMC, and resulting virus outgrowth in the supernatant was used in the TZM-bl assay. No participant maintained full suppression even in the high antibody dose group; however, by 56 days, three of the eight had not yet fully returned to baseline VL (within 0.5 log10). These observations might suggest that virus escape from 3BNC117 occurred but that the virus may not replicate as well.The first passive immunization clinical trial using new-generation bNAbs in humans intravenously infused CD4bs antibody 3BNC117 into 17 viremic subjects (presumably subtype B infected) with a dose escalation between 1 and 30 mg/kg . Overallenv genes for a subset of individuals and Envs were cloned to test for antibody resistance. Overall, cloned Envs from plasma virus became more resistant to 3BNC117 post-infusion, but the number of cloned Envs (three per time point) was low for comparison. Unsurprisingly, the neutralization data from cloned Envs did not exactly match the virus outgrowth neutralization data, highlighting the differences between these two methodologies. When sequences of plasma envs were compared, changes could be detected in some subjects around the 3BNC117 epitope, especially in loop D and loop V5, but there were no consistent resistance mutations detected.To test for virus escape, autologous virus sensitivity to 3BNC117 was tested from pre- and post-infusion time points. Increased resistance to 3BNC117 was detected even in the low-dose group, although the degree to which the virus became more resistant was variable. SGA was performed on pre- and post-infusion plasma virus 10 in six of the eight participants, and mean nadir for all eight was 9 days. Plasma Envs, cloned from before and after infusion, revealed that the disparate effects on VL decline were related to pre-existing virus resistance to VRC01. The two participants with little to no decline in VL had relatively high autologous Env IC80values (geometric mean IC80 of 10 cloned Envs were 30 and 17 μg/ml), and the level of infused antibody was calculated to be less than 100-fold above the mean IC80. Additionally, level of virus suppression appeared to be related to baseline VL because the two participants with the lowest baseline VLs (<1000 copies/ml) maintained virus suppression until very low to undetectable antibody levels were reached. Sequencing and cloning rebound virus from these 2 participants revealed no evidence for increased resistance to or escape from VRC01 suggesting rebound occurred due to low antibody levels [envs revealed changes in the virus quasi-species between pre- and post-infusion. Many of these sequence differences were detected within the VRC01 epitope, especially changes in loop V5 length; however, no consistent resistance mutations were found. Cloned Envs were tested for increased resistance to other bNAbs that may be used in future clinical trials and no difference was detected, apart from slight increase in resistance to the CD4bs antibody 3BNC117.A second phase 1 clinical trial tested infusion of CD4bs bNAb VRC01 into chronically HIV-1-infected individuals and many of the findings were similar to the Caskey study . Importay levels . The othTogether, these two clinical trials indicate that, similar to the NHP model, baseline VL and sensitivity to antibody can affect the degree to which an infused antibody suppresses virus . A more complicated picture was formed in the human trials, where selection pressure against the most sensitive virus species may have been sufficient to allow VL increase in the presence of the antibody. While these reports examined bNAb effects on the virus quasi-species, antibody infusion may have affected the immune response as well.50 or IC80) as is the standard in the field for reporting neutralization data. The use of AUC to quantify neutralization incorporates viruses that are not neutralized up to 50% into the dataset, thereby allowing measurement of subtle shifts in neutralization; however, it also allows measurement of noise within the neutralization assay. Examination of the reported IC50 values with a more stringent cutoff reveals significant differences  in one of 15 infused viremic individuals, none of the infused individuals on ART, and one of the 18 control sera . Within the control individuals, 13 possessed bNAb activity in the pre- and post-infusion samples, which may have confounded this observation. This finding of neutralization improvement in infused participants did not correlate with CD4 level, VL, or pre-infusion neutralization capacity. Longitudinal SGA of the virus quasi-species in a subset of individuals detected inconsistent changes in diversity post-infusion, and confirmed that, similar to previous findings [One reason for use of antibodies as treatment is their potential to exert multiple anti-viral effects. Theoretically, infusion with bNAbs could not only neutralize circulating plasma virions but also bind infected cells for Fc-mediated clearance as well as form immune complexes that could enhance antigen presentation. A follow-up study of the 3BNC117 clinical trial examined the possibility of enhanced immune function following bNAb infusion by analyzing sera and virus 6-month post-infusion, once 3BNC117 was no longer detectable in serum . IgG wasfindings –41, the 50, suggesting that breadth may have been naturally developing. Altogether, observations of modest increases in breadth and potency after infusion with a heterologous antibody, while intriguing, remain to be fully explored and confirmed.The mechanisms behind this phenomenon of augmented neutralization may include increased virus diversification in the presence of heterologous antibody leading to new B cell stimulation or the presence of increased immune complexes that serve as immunogens expanding B cell responses. Interestingly, the virus in subject 2C4 was resistant to 3BNC117 and VL did not decline in the presence of the antibody , but the50 ≤2.0 μg/ml to 3BNC117 were enrolled. The average time to rebound was 8.4 weeks, and this result was significantly different from the 2.6 weeks in matched historical controls, from previous AIDS Clinical Trials Group (ACTG) ATI studies. In this study, time to rebound did not correlate with pre-ATI virus outgrowth sensitivity to 3BNC117, with cell-associated HIV-1 DNA, or with sera concentrations of infused antibody. It is important to note that because all participants were pre-screened for sensitivity to 3BNC117, correlation between pre-infusion sensitivity and time to rebound may not be possible to measure in this analysis. Rather, it is conceivable that once the virus has reached some threshold of sensitivity, differences in time to rebound may be difficult to detect. Interestingly, participant 711 had the shortest time to rebound despite 3BNC117-sensitive virus as measured by IC50 or IC80, but this pre-infusion virus was incompletely neutralized (with a maximum percent inhibition of 95%) at the highest concentration of antibody (50 μg/ml). This observation highlights the importance of in vitro pre-infusion virus sensitivity to clinical outcome during passive therapy, with particular emphasis on incomplete neutralization, a common trait among all bNAb classes [Recent studies of ATI allow assessment of the ability of antibodies to suppress virus when baseline virus is undetectable . A paper published by Scheid et al. describes the results of a phase IIa open label clinical trial where 13 participants, with VL <50 copies/ml for over 12 months, were infused multiple times with antibody 3BNC117 and discontinued ART 2 days after the first infusion . Before  classes .80 of the virus. This observation supports the notion that for certain individuals and perhaps particular viral genetic determinants, monotherapy can suppress virus replication if a certain level of antibody is maintained. SGA and cloning of Envs from plasma during virus rebound in seven participants confirmed that, for the majority of subjects, rebound virus was more resistant to 3BNC117 compared to pre-infusion virus outgrowth culture. It should be noted, however, that comparisons between pre- and post-infusion virus are difficult in low-plasma virus settings. The authors phylogenetically compared the sequences of a few virus outgrowth cultures to rebound plasma virus sequences, because pre-ART plasma samples were unavailable. Despite this limited sampling, it is clear that post-ATI plasma rebound virus was overall monophyletic in the eight participants analyzed. Again, analysis of rebound virus found residue changes within the epitope of 3BNC117 in loop V5 or loop D that could be associated with resistance; however, there was no consistent pattern of change. Rebound viruses were tested for increased resistance to other antibodies that may be used in clinical trials and generally no difference was found.Four subjects, whose virus did not become more resistant to 3BNC117, rebounded in the presence of low antibody concentration. The authors calculated that VL became detectable in these individuals only when antibody concentrations waned to less than 10-fold above the mean ICenv genes from eight participants in the ACTG trial were cloned from both before ART and after ATI time points. Strikingly, in some participants, there was a clear monophyletic rebound virus suggesting genetic restriction in the presence of VRC01, and rebound virus was demonstrated to be more resistant to the antibody in six of these individuals. Interestingly, the two subjects with the longest time to rebound in this study were the two with the most VRC01-sensitve virus before ART, and in most other subjects, pre-existing resistance in virus populations could be detected. The NIH trial measured the neutralization sensitivity of replication-competent viral isolates from autologous CD4 T cells before and after VRC01 infusion, and indeed, this study found increased resistance to VRC01 in six of seven subjects tested, which was significantly different in four.Two subsequent trials (NIH 15-I-0140 and ACTG A5340) tested the ability of VRC01 infusion to delay virus rebound during ATI; however, in these trials, which examined 10 and 13 participants respectively, pre-screening for VRC01-sensitive virus was not performed . The medTogether, these studies highlight the impact of pre-screening virus for in vitro sensitivity as an indicator of greater in vivo clinical effect and certainly suggest that for monotherapy with antibodies, pre-screening may be of greater consequence than for ARV drug resistance. Furthermore, there is evidence for genetic restriction of rebound virus during ATI in the presence of antibody, which although not clinically relevant does indicate viral selection pressure by the infused antibody. Finally, the lack of resistance signatures discovered in these studies may reflect the complex nature of the CD4bs epitope, which may be true for CD4bs antibodies in general.The question of timing of antibody treatment is now being investigated in the SHIV-NHP model where combination antibody treatment during early infection (Fiebig I–II) was demonstrated to decrease VL at the same pace as cART treatment . This stOptimizing methods of antibody delivery are currently being explored, including vectored antibody genes. Adeno-associated virus (AAV) delivery of bNAbs as well as CD4 mimetics has demonstrated protection in animal models, but may not be feasible as a therapeutic strategy in the context of antiretrovirals , 48. IncWhile this review is focused on the neutralization breadth and potency of bNAbs, which is mainly derived from their antigen-binding variable region (Fv), the whole antibody, variable and constant region (Fc) together, can exert anti-viral effects beyond neutralization. Antibodies can tag virions or infected cells for phagocytosis or killing, and these processes involve Fc binding to Fc receptors (FcR) on phagocytic or effector cells. In a setting of chronic HIV infection, passive infusion of bNAbs may greatly facilitate these effector functions that an otherwise exhausted and dysfunctional B cell response may not be capable of. Passive mAb therapy provides an opportunity to engineer and optimize half-life or polyfunctionality through Fc mutations, glycosylation patterns, and the use of particular isotypes as reviewed in , 52. In A role for bNAbs has also been implicated in HIV-1 cure strategies, which have focused on methods to reverse latent virus (“shock”) and to mobilize the anti-HIV-1 immune response (“kill”). Taking antibody engineering one step further, many groups have followed the path of cancer immunotherapy by designing two-armed molecules that combine one HIV-recognizing arm with an arm that can activate effector cells These molecules mainly utilize two formats: bi-specific T cell engagers (BiTEs) and dual-affinity re-targeting (DART) molecules. Both formats have been tested using Fabs of bNAbs as the HIV-specific arm; however, the measurement for these assays is killing of infected cells, not neutralization. One study by Pegu et al. constructed BiTEs using the Fab region of bNAb VRC07-G54W linked to an anti-human CD3 single-chain variable fragment and found that the BiTE could activate and lyse latently infected cell lines . Interes80) rather than absolute (need to be >100 μg/ml), with particular importance on prevalent subtypes in study populations. The clinical trials described here were performed in North America with predominantly subtype B-infected individuals. Data to address differences in subtype sensitivity may result from the current HVTN Antibody Mediated Protection Trials, which will infuse VRC01 into US and African populations which will infuse VRC01 into HIV negative US and African populations for prevention as opposed to HIV therapy.Overall, data in human clinical trials support the observations from NHP models that infusion of heterologous bNAbs during chronic infection can exert significant effects on the virus; however, factors such as potency of neutralization and baseline VL affect the outcome. Pre-screening participants for antibody sensitivity presumably led to increased observed clinical effects (better VL decline in viremic subjects and longer time to rebound during ATI) . While tThe human trials described here have invaluably demonstrated that virus selection occurs in the presence of heterologous antibody and emphasize the need for combination antibody therapy in the future. It will be important to address optimizing bNAb combinations for geographic subtype  and anal


On 1 February 2016, the World Health Organization (WHO) declared that clusters of microcephaly cases and other neurological disorders occurring in Zika virus (ZIKV)-affected areas constituted a public health emergency of international concern. Increased surveillance of the virus, including the requirement for laboratory confirmation of infection, was recommended. The WHO Regional Office for the Western Pacific therefore initiated a rapid survey among national-level public health laboratories in 19 countries and areas to determine regional capacity for ZIKV detection. The survey indicated that 16/19 (84%) countries had capacity for molecular detection of ZIKV while others facilitated testing through referral. These results suggest that robust laboratory capacity is in place to support ZIKV surveillance in the Western Pacific Region. Aedes mosquitoes, the same vector transmitting other arboviruses of public health impact such as yellow fever virus, dengue virus (DENV) and chikungunya virus (CHIKV). . The survey was completed by 23 laboratories in 18 countries. For the country not responding, information from other sources such as recent peer-reviewed publications was used where possible to augment the data set and cover all 19 countries.AMRO, World Health Organization Regional Office for the Americas; CHIKV, chikungunya virus; DENV, dengue virus; PCR, polymerase chain reaction; US CDC, United States Centers for Disease Control and Prevention; ZIKV, Zika virus.Given the similarity of disease presentation, . (As in our previous survey of regional PCR testing capacity for Middle East respiratory syndrome coronavirus, (While the Region seems prepared overall for testing of ZIKV, it is important to continue strengthening the apparatus for detection particularly through ensuring testing proficiency by EQA participation and enhancing referral mechanisms and International Air Transport Association certification where needed. It should be noted that while the EQA for DENV and CHIKV diagnostics gives confidence about regional testing proficiency for those arboviruses, (The laboratory plays an important role in improving our understanding of ZIKV epidemiology. While this survey reveals a broad availability of molecular diagnostics to support surveillance of ZIKV in the Western Pacific Region, further key roles remain for laboratories in helping to unravel the pathogenicity of the virus and its potential causal role in the observed cases of microcephaly and other neurological disorders.


Copy number variation is now recognized as one of the major sources of genetic variation among individuals in natural populations of any species. However, the relevance of these unexpected observations goes beyond diagnosing high diversity.Here, it is argued that the molecular rates of copy number variation, mainly the deletion rate upon variation, determine the evolutionary road of the genome regarding size. Genetic drift will govern this process only if the effective population size is lower than the inverse of the deletion rate. Otherwise, natural selection will do. The advent of genomic systems biology is leading, in very recent years, to the discovery of widespread genetic features, previously unrecognized in complex organisms like humans; they can then have strong implications in biomedicine. One of these fascinating features is copy number variation , which iIn this short piece, it is argued that these rates greatly determine the way by which the genome can increase its size, i.e., the evolutionary force that controls this process. Indeed, the acquisition of genetic redundancy is believed to be the major mechanism to increase genome size, and then genome complexity , 6. For μ denotes the duplication rate of a locus and λ the deletion rate upon duplication, the frequency of the genotype with two copies (x) in a population of size N is governed by the following stochastic differential equationt is measured in generations, and z(t) is a stochastic process with mean zero and correlation delta, having assumed a Wright-Fisher reproduction model and strictly neutral selective conditions  the variance of the stochastic process, x(1 − x)/N, to have constant diffusion, in good tune with the Kimura’s calculation , which yields the condition of λN < 1/12, when λ ≫ μ  [λN < 1 to have chances for fixation. By contrast, if (μ + λ)N ≫ 1, the system is mostly dominated by the balance between duplication and deletion. Therefore, Δx ≪ 1, which entails that duplications cannot be fixed.Fixation will occur in displacements that reach The deletion rate that was considered here is the rate at which a repeated portion of the genome is deleted. Certainly, duplication imposes a genetic instability that is generally resolved by deletion ; sometimSalmonella enterica, μ ≈ 10−4/locus/gen. and λ ≈ 2·10−2/locus/gen. [N ≈ 108. In this case, (μ + λ)N ≈ λN ≈ 2·106 ≫ 1, which entails that this bacterium cannot acquire genetic redundancy, at least by drift. Similar is the case for the lower eukaryote Saccharomyces cerevisiae, where μ ≈ 3·10−6/locus/gen. and λ ≈ 2·10−6/locus/gen. [N ≈ 107, give (μ + λ)N ≈ 50 ≫ 1. However, the scenario is different in higher eukaryotes. For Drosophila melanogaster, μ ≈ 2·10−7/locus/gen. and λ ≈ 10−6/locus/gen. [N ≈ 106. This results in (μ + λ)N ≈ λN ≈ 1, the soft limit, suggesting that transient fixation of duplications could occur. Better is the case for Caenorhabditis elegans, as μ ≈ 10−7/locus/gen. and λ ≈ 2·10−7/locus/gen. [N ≈ 105, lead to (μ + λ)N ≈ 0.03 ≪ 1. For Homo sapiens, μ + λ ≈ 10−6/locus/gen. [N ≈ 104, ensures many momentary fixations by drift, as (μ + λ)N ≈ 0.01 ≪ 1.This simple theory can be applied to analyze the fixation ability in different organisms Fig. . For Salcus/gen. , with N cus/gen. , with N cus/gen. , with N μ and λ) confidently estimated until now (in mutation accumulation experiments) are really scarce, only available for some model organisms [Arabidopsis thaliana, e.g., only bioinformatic estimates have been produced, although these give values that differ from experimental estimates in several orders of magnitude. Based on the values of D. melanogaster and C. elegans, one can predict λ ≈ 10−7–10−6/locus/gen. for A. thaliana, resulting in λN ≈ 0.03–0.3 < 1, as N ≈ 3·105. Higher eukaryotes have indeed more chances to transiently fix duplications by drift due to a reduced effective population size [But the rates .No consideration of high-order variations, such as gene triplications or quadruplications, while these are found in nature. No consideration of individuals with zero copies, assuming they are deleterious and then quickly diluted.Population size assumed constant, while this may vary with time due to multiple environmental factors.The following limitations associated with the mathematical model were identified:


Plant viruses and their virus-like particles (VLPs) have a lot of advantages for biotechnological applications including complete safety for humans. Alternanthera mosaic virus (AltMV) is a potentially promising object for design of novel materials. The 3D structures of AltMV virions and its VLPs were obtained by single particle EM at ~13Å resolution. The comparison of the reconstructions and a trypsin treatment revealed that AltMV CPs possesses a different fold in the presence (virions) and absence of viral RNA (VLPs). For the first time, the structure of morphologically similar virions and virus-like particles based on the coat protein of a helical filamentous plant virus is shown to be different. Despite this, both AltMV virions and VLPs are stable in a wide range of conditions. To provide a large amount of AltMV for biotechnology usage the isolation procedure was modified. Potexvirus, family Alphaflexiviridae. Flexible filamentous virions of AltMV have helical type of symmetry; they are ~570 nm long and ~ 13 nm in diameter. Virus contains a positive single-strand RNA, 6606 nt long. AltMV was for the first time isolated and described in Australia in 1999 [Alternanthera mosaic virus (AltMV) is a plant virus belonging to the genus  in 1999 , the USA in 1999  and Asia in 1999 . AltMV-M in 1999 . AccordiIn contrast to rich information on the rigid rod plant viruses, structural information for flexible plant viruses has been lacking for long time. Now, the high-resolution structures are available for PapMV , bamboo in vitro at pH 4.0 and low ionic strength [Similarly to PapMV CP , AltMV Cstrength . Howeverstrength .Recently, plant viruses and VLPs, derived from them, have been used extensively in biotechnology including the development of medical products (vaccines) –19. PlanThis work is devoted to investigation of the possibility of obtaining AltMV VLPs under different conditions and to a detailed characterization of AltMV virions and VLPs properties in terms of their biotechnological potential. In addition, the method of AltMV isolation and purification was modified, markedly increasing the yield of the purified virus.The initial infected material was received from the German Collection of Microorganisms and Cell Cultures, Braunschweig as a strain of PapMV. We propagated this virus and defined it as a new strain of AltMV–AltMV-MU (Moscow University) (accession number FJ822136 in the GenBank) .Nicotiana benthamiana or Portulaca grandiflora. Plants were provided by the greenhouse of Faculty of Biology, Lomonosov Moscow State University. Plants were inoculated using sap extracts  from plant tissue of infected P. grandiflora. 30 days after infection, the plant biomass was frozen at -20°C. For virus purification, plants were homogenized in 0.3 M glycine-KOH, 1% Na2SO3, pH 7.5 (3 ml buffer per 1 g of leaves) in a blender. All steps of virus preparations were carried out at 4°C and with chilled buffers. The plant sap was then filtered through a cheesecloth, and the filtrate centrifuged at 12,000 rpm for 30 min . The supernatant was collected and clarified with Triton X-100 added to 1% (v/v) and incubated for 20 min. Polyethylene glycol  and NaCl were added to the solution to 5% and to 2% (w/v), respectively. After dissolving the components, the solution was kept overnight at +4°C. The PEG precipitate was collected by centrifugation at 10,000 rpm for 20 min . The sediment was collected and retained at +4°C. The PEG (Mr 6000) was added to the supernatant to a final concentration of 8% (taking into account the one added earlier). After dissolving the components, the solution was kept overnight at +4°C. The PEG precipitate was collected by centrifugation at 10,000 rpm for 20 min  and combined with the virus precipitate obtained on the previous day.The virus was maintained in either Extraction of the obtained precipitate was carried out two to three times for 2–6 hours by gentle shaking (until the precipitate was completely dissolved) with a buffer containing 0.05 M Tris-HC1, 0.01 M EDTA, pH 8.0. The resulting extract was centrifugated at 10,000 rpm for 20 min . The virus suspension was then centrifuged at 110 000 g for 3 h . The pellets were suspended overnight at 4°C in 0.05 M Tris-HC1, 0.01 M EDTA, pH 8.0 and clarified by centrifugation at 10,000 g for 20 min. Then 8 ml of the virus suspension per 10 ml S303922A - 10PC BOTTLE (B) tube was underlaid with 2 ml of 30% sucrose . Samples were centrifugated at 110,000 g for 4 h . The pellets were resuspended in 0.025 M Tris-HCl buffer, pH 8.0; 0.01 M Tris-HCl, pH 7.5 or water. The resulting virus solution was centrifugated at 10,000 g for 20 min. Virion concentration was estimated as described [The method of salt deproteinization with 2M LiCl as described previously ,24.The AltMV virions and virus-like particles were incubated at room temperature for 1 hour under different conditions . Results were analyzed by electron microscopy.The samples were prepared as described by Nikitin et al.  and exam−/Å2) were collected on a Tecnai G12 Spirit Twin electron microscope  operated at 120 kV, using a 4x4 Eagle CCD camera . A defocus range between 0.8 and 3.5 μm underfocus was used.The AltMV samples were diluted to a final concentration of 1.5mg/ml in 25mM Tris-HCl, pH 7.5. 3.5 μl of the AltMV was applied to glow-discharged Quantifoil 1.2/1.3 grid . The grids were then blotted and frozen in liquid ethane using Vitrobot . Low dose images  and the number of subunits (from 8 to 10 Å in increments of 0.01 Å) per rotation of the helix. As a result, 50 reconstructions were generated. The pitch of the helix was estimated using 50 best reconstructions based on the average cross-correlation index and amounted to 35.7 Å. The determination of the number of subunits per helix is based on an analysis of the amplitude of the correlation between the class-average and the re-projections of the 3D models. The subsequent improvement of the reconstruction was carried out in 50 iterations, until the resolution of the electron density map did not cease to improve.The resolution of the final 3D reconstructions was estimated by the Fourier shell correlation (FSC) using a 0.143 criterion. This was done by splitting the final average and symmetry related views into even and odd numbers. The reconstructions from the two halves of the data were used in the FSC calculation.To generate unbiased difference maps, the 3D reconstructions of AltMV virions and VLPs were aligned against their symmetry axis using Chimera 1.9  before sThe trypsin  was added to the samples, and they were incubated in 100 mM Tris-HCl, pH 8.0 for one hour at 37°C as described in .For the successful use of plant viruses in biotechnologies, it is necessary to provide the effective accumulation, isolation and purification of the viral particles. The high yield of virus per unit of plant biomass is important to the economic expediency of plant viruses’ usage. Undoubtedly, among plant viruses actively used in biotechnology, the Tobacco mosaic virus remains the "champion" in accumulation in plants: its yield reaches up to 10 g / 1 kg of green plant biomass .Chenopodium amaranticolor [Nicotiana benthamiana [Portulaca grandiflora [P. grandiflora and N. benthamiana. Geering and Thomas [C. amaranticolor, yielding a 23.4 mg of virus per 100 g of infected leaves. This is a quite high yield, but the host plant used for this cannot be considered optimal for the accumulation of AltMV. C. amaranticolor is the host for a wide variety of plant viruses, including the representatives of the Potexvirus genus that can lead to contamination of the AltMV with other viruses [P. grandiflora is hardly susceptible to infection with other viruses [N. benthamiana is a classical laboratory plant very convenient for work. We conducted a passage of infectious material through P. grandiflora for obtaining a pure preparation of AltMV to ensure that no coinfection occurs.The first stage in this work was the optimization of the AltMV purification protocol to produce large quantities of virus. Earlier, AltMV was isolated from nticolor , Nicotiathamiana  and Portndiflora ,15. In o viruses . Therefo viruses , and N. AltMV was purified following a procedure initially developed for another potexvirus, Potato virus X, with some modifications ,15. GeerBased on the method described in Mukhamedzhanova et al.  and IvanImages of AltMV viral particles were obtained using electron microscopy (EM), both in negative stain  and in cUsing 230 viral segments cut from raw EM images  the 3D mThe stability of viral particles in different conditions, especially in conditions close to physiological, is an important factor for their successful use in biotechnology. Analysis by electron microscopy showed that the AltMV virions retain their overall morphology and are stable after incubating them for 1 hour in distilled water , in 0.15in vitro in the absence of RNA with the formation of VLPs not only at acidic pH 4.0, but also at pH 8.0, which significantly distinguishes it from PapMV CP [As it was shown previously, AltMV CP is able to polymerize PapMV CP ,15. In tUsing single particle EM, we studied VLPs both in negative stain  and in cAccording to our recent data, native AltMV-MU virions and VLPs are antigenically related, but not identical . This inHere, we demonstrated that upon trypsinolysis the CPs of AltMV virions remained intact , lane 2 The VLPs are stable under the same conditions as the native AltMV virions. VLPs do not change their morphology and size during incubation in distilled water , in 0.15in vitro into RNA-free VLPs under various conditions, including physiological pH 7.5 and in the presence of NaCl, as well as in the serum of laboratory animals . Adding of 0.2 M NaCl at pH 4.0 results in the assembly of much shorter VLPs ,38. In c animals .Undoubted interest in plant viruses, virus-like particles (VLPs) and the possibility of their use in the development of new biotechnologies  is constantly growing. Obtaining of VLPs under various conditions, and study of the structure, properties and stability of virions and VLPs is a direction of research of great significance. The economically expedient use of viruses in biotechnology is possible only if they are produced in large quantities. Therefore, in this work the procedure for the Alternanthera mosaic virus (AltMV) isolation was modified. As a result, the yield of AltMV was significantly increased (up to 57.3 mg of virus/100 g of plant biomass).Here, for the first time, the 3D structures of AltMV virions and its VLPs were obtained by single particle EM at ~13Å resolution. The comparison of the reconstructions revealed that AltMV CPs might have a different fold in the presence and absence of viral RNA. This has been confirmed by a trypsin hydrolysis. Thus, for the first time the structure of morphologically similar virions and virus-like particles based on the coat protein of a helical filamentous plant virus is shown to be different.in vitro stable extended polymers–RNA-free VLPs–under different conditions; VLPs are identical in morphology, but differ in structure to the AltMV virions. The principal difference between VLPs of AltMV and VLPs of the related papaya mosaic virus is the high stability of AltMV VLPs in a wider range of conditions, including physiological, and the absence of nucleic acid in their composition.AltMV virions are demonstrated to be stable in various conditions, including the serum of laboratory animals. AltMV coat protein (CP) is shown to be able to form S1 FigA. AltMV virions, B. AltMV VLPs. Top row–segments, cut from raw image; below–corresponding class averages. Right–width determination of the helical particle.(TIF)Click here for additional data file.S2 FigA. in distilled water, B. in 0.15 M NaCl, C. in 0.01 M Tris-HCl, 0.15 M NaCl, pH 7.5, D. in mouse serum. Incubation for 1 hour. TEM, staining with 2% uranyl acetate.(TIF)Click here for additional data file.S3 FigA. AltMV virions, B. AltMV virions treated by trypsin, C. AltMV VLPs, D. AltMV VLPs treated by trypsin. Incubation with the enzyme for 1 hour. TEM, staining with 2% uranyl acetate.(TIF)Click here for additional data file.S4 FigA. in distilled water, B. in 0.15 M NaCl, C. in 0.01 M Tris-HCl, 0.15 M NaCl, pH 7.5, D. in mouse serum. Incubation for 1 hour. TEM, staining with 2% uranyl acetate.(TIF)Click here for additional data file.


Parkinson's disease (PD) is the second most prevalent neurodegenerative disease worldwide, affecting more than four million people. Typically, it affects individuals above 45, when they are still productive, compromising both aging and quality of life. Therefore, the cost of the disease must be identified, so that the use of resources can be rational and efficient. Additionally, in Brazil, there is a lack of research on the costs of neurodegenerative diseases, such as PD, a gap addressed in this study. This systematic review critically addresses the various methodologies used in original research around the world in the last decade on the subject, showing that costs are hardly comparable. Nonetheless, the economic and social impacts are implicit, and important information for public health agents is provided. Health and the economy are related intrinsically. The purpose of the studies on the costs of diseases is describing them, estimating costs, comparing established programs, and projecting these costs based on clinical, demographic, epidemiological, and technological factors. In fact, over the past decade, there has been a growing number of studies, which are presumed to be valuable decision tools, because the limited amount of resources must be used rationally and efficiently as not to miss opportunities to improve overall population health .In neurodegenerative diseases, such as Parkinson's disease (PD), whose prevention is still impossible, the burden borne by society, whether it is financial, social, or even psychological, is often heavy. Being the second most prevalent neurodegenerative disease worldwide, it generally affects individuals between 40 and 50 (late-onset PD) , 3, compStudies on disease costs may have several approaches, such as economic assessment, epidemiological design, or even type of cost involved, as well as the viewpoint of defining resource use strategies. The diversity in methods is a significant factor why cost estimates differ between studies, opening the discussion about which public policies are most appropriate for PD.This systematic review provides introductory concepts on the types of studies on costs and analyzes results in selected articles critically, highlighting the benefits and limitations of their methods. Moreover, this study identifies the most common studies regarding DP costs worldwide over the past 10 years, showing possibilities for studies being carried out in Brazil, where there is a lack of this type of analysis because most studies only involve the clinical aspects of the disease. online bibliographic information services were accessed—SCOPUS and PubMed—with the aim of selecting original articles about the cost of PD over the past decade.In March 2016, two (parkinson disease) AND TITLE-ABS-KEY (economics) OR TITLE-ABS-KEY (costs of illness) OR TITLE-ABS-KEY  OR TITLE-ABS-KEY  OR TITLE-ABS-KEY  OR TITLE-ABS - CUT  OR TITLE-ABS-KEY  OR TITLE-ABS-KEY (direct costs) OR TITLE-ABS-KEY (CB costs) OR TITLE-ABS-KEY (out of pockets) AND DOCTYPE (air OR re) AND PUBYEAR > 2004 AND ) AND ).The following terms were used for access: abstract. Although 35 of these had a suggestive title, that is, they did not tackle general costs of disease exclusively, 107 were separated for complete reading, and 30 met the criteria for research . Once the first levels of inclusion were satisfied, 142 papers were selected for reading theresearch .The determination of the costs of a disease facilitates learning what its burden to society is, assessing its degree of efficiency, and understanding how the market tends to organize itself regarding certain values .The basic function of any economic assessment is to identify, measure, value, and compare the costs and consequences of alternative proposals –8.In this case, four techniques are possible :(1) Cost-Minimization. Cost-minimization is the least used technique, because it only compares costs of interventions that produce the same outcomes with different costs. For chronic diseases, such as PD, there are no studies using this type of analysis.(2) Cost-Effectiveness. Cost-effectiveness is the technique most used in literature, which assesses the impact of different alternatives that bring better results with lower costs; these are always comparative and explicit and designed to select the best option to achieve what is perceived in clinical practice.(3) Cost-Benefit. This analysis determines whether a new health technology or intervention generates net benefits to society. However, due to its difficulty, complexity, and controversies in valuing human life and certain health conditions in monetary terms, this analysis is rarely found in the literature.(4) Cost-Utility. This analysis assesses the impacts on survival and quality of life, which are determining criteria to judge the effects of strategies in health care, that is, the level of well-being and preferences of the individual.The epidemiological study designs define how the research will be performed in relation to adopted method –13. The (1) Prevalence and Incidence. The prevalence estimates the number of deaths, hospitalizations, prevention, and research attributable to a disease in a given period , to subsequently estimate the costs incurred by these consequences. Incidence refers to the number of new cases in a predefined period, and it foresees the associated costs from the onset of the disease until its disappearance , through a rough projection of the flow of these values.Studies on prevalence display greater results than incidence ones, for diseases are usually long-term sequelae, as is the case of PD, and they are of great importance in planning specific policies on certain diseases when their economic burden was underestimated. Therefore, they can identify the main components of current expenses and uncharged resources.(2) Top-Down and Bottom-Up.  Top-down approaches are normally used in prevalence studies, when the expenses of a disease are widely known from national or regional statistics. In bottom-up studies, cost estimates are more detailed. The data depend on the scope of the study, and they are intrinsically related to the unit costs of inputs used, through interviews, questionnaires or chart review, and assessment of individual cost. The average cost per person is then obtained by means of the number of times the service was used and the number of people with the disease. Although bottom-up studies are more complete when it comes to resources and more precise regarding patient selection, they run a high risk of double counting costs . The majority of studies with PD adopt this approach.(3) Prospective and Retrospective Approaches. There is a temporal relationship, where in prospective studies the relevant events have not happened yet, that is, studying the patient over time, formalizing a system of data collection focused on the purpose of the research, such as questionnaires designed specifically for patients and/or their caregivers, where everything is recorded in “real time.” In retrospective studies, all events had already occurred when the study was initiated. They are usually employed in long-term chronic diseases, as is the case of PD. In this case, research efficiency can only be possible with enough observational datasets. It would be best if the data were stored electronically to minimize memory bias due to omission of facts or values.(4) Econometric Approaches. Econometric approaches estimate differences between groups. One of the groups has the disease and the other does not; however, both have the same characteristics, which are assessed by several regression analyses involving demographic factors such as sex, age, marital status, ethnicity, relationship between patient and caregiver, housing, and duration of the disease.(5) Markov Models. Markov models are used in several studies of chronic diseases, when patients are studied over time, and they are stratified according to disease scale. In the case of PD, the scale of Hoehn and Yahr (it assesses the degree of disability due to the disease in scores) is key to building this model. These are typically prospective studies, proposing cost increases with disease severity. bottom-up study, for instance, because its purpose, most of the time, is to maximize the content of information, contributing to enriching knowledge.Several approaches may be featured in the same study; that is, we may have a retrospective, prevalent, andThe costs of a disease are typically stratified as follows , 10, 14 (1) Direct Costs. Direct costs are related to the disease and its equation; their charges may concern public administration, insurance companies, the patient, the patient's family, or even a combination of all or some of these determinants. The estimates of direct costs associated with chronic diseases are higher than those associated with acute and communicable diseases, on the condition that better treatments and methods of prevention are adopted. This group can be divided into direct medical costs and nonmedical ones, although not all studies adopt this division.(2) Indirect Costs. Indirect costs refer to the loss of income and/or productivity; they are caused by disease. Additionally, they can incur costs to both the patient and the employer. Depending on the disease, this loss may be partial, temporary, or permanent, and it may be restricted to the patient and/or caregiver (as in the case of advanced stage PD), frequently leading to early retirement. If there is a possibility of returning to regular activities, this disease may not occur on the same productivity level as before, or lead to frequent absences (absenteeism), incurring additional costs, such as loss of promotions.(3) Intangible Costs. Intangible costs are virtually impossible to measure, since they incur psychological and psychosocial costs imposed to the patients, their family, and acquaintances due to the disease, as well as pain, behavioral changes, and everyday activities. They depend on the perception that the patient's health problems lead to social consequences, such as isolation.(4) Personal Costs. Personal costs are the costs borne by the patient and/or their family and friends due to consultations with health professionals, medication, laboratory tests, domestic adjustments, locomotion resources, and the need for home care. Depending on the country, these costs, also called copayments, are borne by the government, health insurance, or religious or private health institutions. Sometimes, these payments are also designated as direct costs because they are associated with the disease. They may be redeemable or not, implying an additional expense to the patient, who has to spend a certain amount of money in advance.Who bears the costs related to the program defines which costs are included for analysis , 7, 10 .(1) Industry . The industry bears the costs due to absenteeism or loss of productivity, and early retirement due to the disease.(2) Society. A more comprehensive perspective considers all costs related to the program, regardless of who will pay the expenses . This approach is thought to be the most appropriate to support health-related decisions. Most research on PD addresses this perspective, although some studies include more than one viewpoint.(3) Patients and Their Family. Costs are borne by the patient concerning appointments, transport used for his treatment, purchase of medication, expenses with caregivers, domestic changes, and so on.(4) Programs, Public Health, and/or Insurance Companies. When there is a need to identify all the inputs related to the disease, for which monetary value explains the base period and the form of assessment used should be assigned, this perspective is highly likely to underestimate the cost of disease, especially when greater profit or lower production costs are targeted.Although PD affects more than four million people worldwide , little PD was considered among the most prevalent and costly diseases of the brain, being the fourth most expensive second study in 28 European countries . HoweverOver the past decade, there has been a significant increase in the number of papers related to costs of diseases. The papers selected for this review are summarized in Practically, all articles used the general costs of the disease, without naming the type of economic evaluation. Only one article  referredBecause there is no way of implementing measures so as to reduce new cases, the most appropriate model for PD costs may be developed from prevalence studies. They are conducted when diagnosis has already been established, obtaining ample results, and are to conduct than incidence ones, which demand rigorous criteria for diagnosis. In this review, we identified 20 papers that followed this line of research see .The use of questionnaires, suggesting a bottom-up approach, is common practice found in the research reviewed here, although not all of them described the design. The unit value of the inputs used is more easily acquired than full reports obtained from large databases in top-down approaches, although at least six papers suggest the use of this approach for studying large samples –27.Since it is a disease with long survival, retrospective studies are the most common for PD, despite the bias of memory that can be generated depending on the retroactive period. In reviewed articles, 12 authors see  opted foThe aforementioned chosen variables are related to the purpose of the study, but, in general, we observed that 20 see  out of 3Among direct costs, the most common variables analyzed in most studies were medication, hospitalizations, outpatient visits, auxiliary treatments, home care, transport, and special equipment. Not all studies divided the direct costs into medical and nonmedical . In one Regarding indirect costs, most studies are related to the patient and/or the caregiver in terms of loss of productivity, early retirement, and sick leaves . As for personal costs, they consider informal care, copayment treatments, drugs, and equipment.The society costs are the most studied, the society being the most affected regarding allocation of resources. Only four studies assessed this prospect from the patient's point of view , 32–34.Clarity is needed in the way data and/or results are expressed, which may generate uncertainty or confusion in the conclusion of a study. For instance, one of the studies  does notMany authors have chosen to direct certain types of costs towards one category, which reinforces the uniqueness of each study. One author  argued tThe fact that the same cost component is classified in different categories can have a strong influence on the final results, not considering the values set by the inputs in each country. In a study conducted in Russia , for exaIssues related to health insurance also influence the comparison of studies greatly and must be considered. For example, in India, a study  revealedn = 12) [n = 630,000) [There are some other factors that certainly affect the results obtained: samples ranged from small cohorts (n = 12)  to large630,000) , some st630,000) , 42 have630,000) , 43 asseClinical, social, demographic, and economic issues of the patient;Medical and nonmedical direct costs;Indirect costs;Personal costs (including caregivers).Finally, with regard to the revised articles of this manuscript, we could suggest an instrument as a guideline to determine PD-related costs even though several methodologies and different variables could be taken into account for each particular scenario. Therefore, prospective studies would be the ideal methodology, but cross-sectional, retrospective ones, with a bottom-up approach from the perspective of society, could be more feasible. The questionnaire to obtain data could be divided into the following parts:The most common variables found in the literature used to determine the costs of PD, depending on the scope of the study, are shown in The concepts mentioned in this review do not aim to finalize the discussion on health economics tackling the costs of neurodegenerative diseases, such as PD, but only to allow access to introductory concepts of these assessments, so that the reader can contextualize the articles analyzed here.The very definition of studies on costs of disease suggests limitations, as the articles here reviewed display methodological heterogeneity regarding PD costs, and this variation is an important factor that should receive more attention in literature. Unlike Alzheimer's disease, which has a validated instrument to determine the costs of illness , PD presWe observed that there is no standardization of terminology used for the definition of costs, or even unanimity in the identification of categories because a variable can be found in different classifications, depending on the criterion used by the researcher, which may underestimate the total cost of the disease.On the other hand, there is no one perfect research methodology covering a single answer for all solutions. Sometimes, certain types of studies are more appropriate than others. There are several limitations that must be discussed and related to, such as the methodological problems, and the validity of their assumptions can differ because they may introduce bias in analysis in favor of a variable, lack of interest in its assessment, or even lack of information. Therefore, researchers must be careful with the source of their data and the method used for performing the calculations, so that their research can be replicated and validated, since there is no specific instrument for the assessment of PD costs. A limitation also deals with the funding sources of studies, which may be from government sources, insurance companies, or pharmaceutical industries, for example, generating important biases that also need to be addressed. It is necessary to define useful metrics for public health and private ones for managers of health, employers, insurance companies, and even patients themselves, because, without this agreement, the work of researchers and the funds invested shall remain uncertain and inconsistent.Nevertheless, if the evidence obtained is of good quality in terms of transparency, there is quality and credibility in the data completeness of the documentation. Overall, if they are relevant to health care , these sThe age group that the PD affects, if well attended to, can experience “healthy aging,” with a good quality of life and preserve its autonomy for longer, thus reducing its cost to the state and society.


We report a case of Zika virus infection that was imported to Japan by a traveler returning from Vietnam. We detected Zika virus RNA in the patient’s saliva, urine, and whole blood. In the Zika virus strain isolated from the urine, we found clearly smaller plaques than in previous strains. Zika virus has been documented in Southeast Asia since the 1940s; however, the prevalence and geographic extent of Zika virus disease in Asia remain unclear (t] 32.0), saliva (Ct 39.1), and whole blood (Ct 38.1) samples. However, we did not detect Zika virus RNA in the serum or semen samples. We diagnosed Zika virus infection in this patient; his symptoms resolved without treatment within 7 days after he initially sought care. A 40-year-old man came to the National Center for Global Health and Medicine in Tokyo, Japan, in the middle of November 2016 with fever and a rash. In early November 2016, he had traveled to Ho Chi Minh City, Vietnam, where he stayed for 10 days. During his return to Japan, he developed fever and a diffuse rash on his face, trunk, arms, and legs. He went to the hospital on the day after his return and reported having been bitten by mosquitoes in Ho Chi Minh City. Upon arrival at the hospital, he had no fever (temperature 36.8°C); physical examination revealed conjunctivitis and a maculopapular rash on his face, trunk, and extremities. Results of laboratory tests showed leukopenia  and a platelet count within reference range. We performed a rapid dengue test ; results were negative for nonstructural protein 1, IgM, and IgG. We performed real-time reverse transcription PCR (RT-PCR) amplification using Zika virus primers and probes with urine, saliva, whole blood, serum (obtained 4 days after symptom onset), and semen samples (obtained 6 days after symptom onset). We detected Zika virus RNA in the urine (cycle threshold [Chttps://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that the isolate was an Asian lineage virus .We successfully isolated the infectious Zika virus from the urine specimen. We amplified the nearly complete genome  of the Zika virus from the isolate using RT-PCR and subsequently sequenced it. BLAST analysis , and the strain we isolated belonged to the Southeast Asian clade. A previous study indicated that a strain isolated in Singapore in 2016  also belonged to the Southeast Asian branch, and it was distinct from the isolates obtained in the Americas (In conclusion, the replicative ability of Zika virus might differ by region and thus influences endemic potential. Further studies are necessary to validate these findings.Comparison of plaque morphology between 2 Asian lineage strains of Zika virus, ZIKV/Hu/S36/Chiba/2016 and ZIKV/Hu/NIID123/2016.


Eidolon helvum) in Africa. Here we compared the susceptibility of these bats to three strains of Lagos bat virus  by intracranial inoculation. To allow comparison between strains, we ensured the same titer of virus was inoculated in the same location of the brain of each bat. All bats (n = 3 per strain) were infected, and developed neurological signs, and fatal meningoencephalitis with lyssavirus antigen expression in neurons. There were three main differences among the groups. First, time to death was substantially shorter in the Senegal and Ghana groups (4 to 6 days) than in the Nigeria group (8 days). Second, each virus strain produced a distinct clinical syndrome. Third, the spread of virus to peripheral tissues, tested by hemi-nested reverse transcriptase PCR, was frequent (3 of 3 bats) and widespread (8 to 10 tissues positive of 11 tissues examined) in the Ghana group, was frequent and less widespread in the Senegal group , and was rare and restricted in the Nigeria group . Centrifugal spread of virus from brain to tissue of excretion in the oral cavity is required to enable lyssavirus transmission. Therefore, the Senegal and Ghana strains seem most suitable for further pathogenesis, and for transmission, studies in the straw-colored fruit bat.Rabies is a fatal neurologic disease caused by lyssavirus infection. People are infected through contact with infected animals. The relative increase of human rabies acquired from bats calls for a better understanding of lyssavirus infections in their natural hosts. So far, there is no experimental model that mimics natural lyssavirus infection in the reservoir bat species. Lagos bat virus is a lyssavirus that is endemic in straw-colored fruit bats  for their ability to mimic a natural infection. We used intracranial inoculation to ensure infection of the brain. All three strains infected brain neurons, resulting in fatal neurologic disease, however only two of the strains showed the ability to reach the site of excretion—the mouth—and were considered a suitable virus to use for further studies of this disease in bats. Lyssavirus genus, in the family of Rhabdoviridae of the order Mononegavirales. Rabies is predominantly transmitted by carnivores, in particular dogs, and causes more than 59,000 human fatalities annually [Rabies is an almost invariably fatal disease caused by rabies virus (RABV) or other members of the annually . As terrannually –4. Additannually . Singulaannually –13. Despannually  and how,Some aspects of the pathogenesis of lyssavirus infections in bats are known. This knowledge is mainly based on experimental infections with four lyssaviruses in natural bat reservoir hosts: rabies virus, Australian bat lyssavirus, and European bat lyssaviruses 1 and 2 . These eOther aspects of the pathogenesis of lyssaviruses in bats are poorly understood, such as mechanisms of virus excretion and transmission, the nature and duration of clinical signs, and the duration of virus excretion. An important limiting factor in this pathogenesis research is that oral excretion of virus is an uncommon event in experimentally infected bats –41.Eidolon helvum). Lagos bat virus, which is divided into four lineages, A to D [www.iucnredlist.org). Infections of LBV in mammals other than bats have been reported sporadically [This study was an initial step to reach our overall goal of developing an experimental model that mimics natural lyssavirus infection in a natural reservoir bat host. With ‘natural reservoir host’ we mean a host that is naturally infected and has co-evolved with the pathogen. For the experimental model, we chose Lagos bat virus (LBV) and the straw-colored fruit bat  and the Institutional Review Board of Noguchi Memorial Institute for Medical Research, University of Ghana, Legon.Bats were anesthetized with a mixture of ketamine  and medetomidine . Bats were euthanized by exsanguination under anesthesia with ketamine (5 mg/kg bw) and medetomidine (0.05 mg/kg bw), followed by cervical dislocation.7.25 median tissue culture infectious dose (TCID50) per ml. The second virus strain was a lineage B LBV isolated from a bat in Lagos Island, Nigeria in 1956 [5 TCID50 per ml. The third virus strain was a lineage A LBV isolated from a bat in Kumasi, Ghana in 2013 [5.7 TCID50 per ml. All viruses were diluted to obtain a final inoculum dose of 103.5 TCID50 in 30 μl.Virus stocks of three LBV strains were prepared and titrated according to standard methods.  The firs in 1956 . It had  in 2013 , and wasWe inoculated each of the three available LBV strains into straw-colored fruit bats in order to find the most suitable virus strain for the development of a model for LBV infection in a natural reservoir host. Although not a natural route of infection, we chose intracranial inoculation because this is the most reliable method to infect the brain, by circumventing the need for the virus to spread from a peripheral inoculation site  to the brain. If we initially used peripheral inoculation and no CNS infection occurred, we would not be able to rule out that the virus was defective. Intracranial inoculation was performed by stereotactic surgery to minimize variation in the site of inoculation and to minimize damage to the brain. The course of infection in inoculated bats was followed until general paralysis was reached in order to provide maximum time for the virus to spread from brain to peripheral sites, including sites(s) of excretion, and to determine whether bats were able to survive infection.Bats were obtained from a captive breeding colony that is maintained in Ghana  that hadBats were anesthetized and their heads were placed in a ‘U’ frame stereotactic instrument . For pain relief and prevention of secondary bacterial infections, each bat was given a subcutaneous injection of butorphanol (0.2–2 mg/kg bw), buprenorphine (0.05 mg/kg bw), metacam  and enrofloxacin . The cranium was exposed by skin incision and application of skin retractors. A 0.8 mm diameter dental drill was used to make a perforation in the parietal bone, unilaterally, 2 mm to the right of the midline, level with the lateral canthi of the eyes . The inoculum (see above) was injected at a depth of 5 mm, in a volume of 30 μl using a Hamilton syringe with a 36-gauge needle at a speed of 5 μl/min. After injection, the needle was kept in place for an additional 4 min before slow withdrawal. The skin was stitched back in the original position. A transponder chip for individual identification and for measurement of body temperature was placed subcutaneously in the interscapular region (Bio Medic Data system Inc.). Anesthesia was reversed with atipamezole . Bats were given oral enrofloxacin (0.6 ml/kg bw/day) and metacam (0.1 mg/kg bw/day) for three days after the day of the surgery.Bats were initially observed twice daily, at 07.00 and at 16.00 GMT for the presence of clinical signs. After the first occurrence of clinical signs, frequency of observations was increased to every two hours, day and night. Observers were unaware of the group the bat was in. Oral swabs  were taken twice per week until the onset of clinical signs, when they were taken daily until death. This resulted in oral swabs being taken one to three times per bat in the period of four days prior to death. Oral swabs were collected in RNAlater (Ambion) for viral RNA detection. Euthanasia was delayed to a time point of severe clinical signs  in order to determine whether bats could survive LBV infection. The mock-inoculated bats were euthanized after the last virus-infected bat had died (day 9 post inoculation).Necropsies and tissue sampling were performed according to a standard protocol as soon as possible (approximately 1 to 6 hours after death) within a class 2 biological safety cabinet in a biosafety level 3 laboratory. At necropsy, a standard range of tissues (see below) was collected (1) fixed in neutral-buffered 10% formalin for histological examination, (2) in plain dry tubes for virus isolation, and (3) in tubes with RNAlater for RNA extraction. The following 14 tissue samples were collected: brain , and arbitrarily selected pieces of salivary gland, tongue, heart, lung, liver, kidney, spleen, submandibular lymph node, duodenum, jejunum and colon. Each tissue was collected using a new pair of disposable forceps and a new scalpel blade on an individual gauze pad to prevent possible cross-contamination. For the tissues for histological examination, the formalin was replaced once after two to three days to enhance fixation; the samples were stored at room temperature. The samples in RNAlater were allowed to fix overnight at 4 °C, and then stored at -70 °C until analysis, whereas plain samples in dry tubes were flash-frozen at -70°C.A range of tests were performed in biosafety level 3 laboratories at the Animal and Plant Health Laboratory Agency, U.K. and at the Friedrich Loeffler Institute, Germany, as described below:Hemi-nested reverse transcription-PCR. All tissues and oral swabs, were tested with hemi-nested reverse transcription PCR (hn-RT-PCR) according to a previously-described protocol [protocol . This teprotocol , 59.Fluorescent antibody test Touch impressions on glass microscope slides of the three different parts of frozen brain were made and stained with fluorescein-isothiocyanate (FITC)-conjugated anti-rabies mouse monoclonal antibody  [ anti-N) . A rabieThe formalin-fixed tissues were embedded in paraffin wax, cut in 4-μm-thick serial sections and routinely stained with hematoxylin and eosin to detect microscopic lesions. Immunohistochemistry was performed to detect lyssaviral antigen . BrieflyBats in the Ghana and Senegal groups died or had to be euthanized earlier (four to six days after inoculation) than bats in the Nigeria group (8 days after inoculation) . The timAll parts of the brain  of all virus-inoculated bats tested positive by both hn-RT-PCR and fluorescent antibody test for lyssavirus RNA and antigen respectively, while the brains of all mock-inoculated bats tested negative. The spread of lyssavirus to organs outside the brain , based on hn-RT-PCR testing of 11 extra-encephalic tissues per bat, differed substantially among groups . In the All oral swabs tested negative by hn-RT-PCR. The virus was detected by hn-RT-PCR in salivary gland in all bats from the Senegal group, and in one bat from the Ghana group. However, virus was detected in just one salivary gland with immunohistochemistry, and this was in a bat’s salivary gland that had tested negative with hn-RT-PCR (see below).At necropsy, no gross lesions were observed in any of the virus-inoculated or mock-inoculated bats. On histopathological examination, all virus-inoculated bats had diffuse, mild to moderate meningoencephalitis. This was characterized by the presence of a few to moderate number of lymphocytes in the meninges, around blood vessels . In the There were a few lesions in bats 6, 8 and 11, which we considered to be incidental and not directly caused by LBV infection. Bat 6  had an acute, mild, multifocal fibrinopurulent pneumonia, characteristic of aspiration pneumonia. This bat was noted to have had paresis and muscle spasms during the terminal 24 hours of infection, which could have caused aspiration of water. This lesion, therefore, might have been indirectly caused by the LBV infection. Both bat 6 and bat 8 (Nigeria group) had a chronic, mild, multifocal lymphoplasmacytic interstitial nephritis. Bat 8 had an acute mild focal necropurulent hepatitis. Bat 11 (Ghana group) had deeply eosinophilic, homogeneous to laminated, irregularly shaped structures  in the lumina of about half of the secretory ducts of the salivary gland. Occasionally these structures compressed the lining ductular epithelial cells. None of the mock-inoculated bats had histological lesions in any of the tissues examined, including the brain.p = 0.004, paired t-test; Cells positive for immunohistochemical staining, and thus positive for lyssavirus antigen, were found in a number of tissues. In most of these tissues, positively-stained cells could be clearly identified as neurons based on their morphology. When detected in neurons, antigen was located in the cytoplasm, with staining consisting of small (pinpoint to approximately 2 μm diameter) or large (approximately 5 μm diameter) granules. The number of granules per cell ranged from one to numerous. Neurons with multiple antigen granules were often found adjacent to neurons that did not contain any staining. Variation in the size and number of granules per neuron differed among groups. Stained neurons in the Senegal and Ghana group bats contained only small granules, with a high variation in the number of granules per neuron. In contrast, stained neurons in the Nigeria group bats contained large granules with little variation in the number of granules per neuron . OverallIn the brain, antigen-positive neurons were generally concentrated in the most caudal brain section examined, otherwise there were no clear patterns within or between groups comparing percentage of neurons infected across the six brain sections examined for each bat . We coulIn the extra-encephalic tissues examined, lyssavirus antigen was detected in neurons. Antigen-positive neurons were found only in ganglia in salivary gland, tongue, and heart, and in myenteric plexi  in duodenum and jejunum ; Table 4In tongue sections, lyssaviral antigen was detected not only in neurons but also in epithelial cells. Three bats had one to three foci of lyssavirus antigen-positive epithelial cells at the tongue surface . In bat Antigen was not detected in salivary gland epithelial cells of any virus-infected bat, although salivary gland sections were available for all nine. Antigen was not detected in any other tissues of the virus-infected bats, and was not detected in any of the tissues examined from the mock-inoculated bats.Our results show that all three LBV strains are capable of infecting and replicating in the brain of the straw-colored fruit bat following intracranial inoculation. The predominant detection of virus in neurons confirms that LBV is neurotropic, similar to RABV and other lyssaviruses in other host species . In manyVirus spread from the site of intracranial inoculation to peripheral tissues was detected most frequently in bats infected with the Senegal and Ghana strains of LBV. The rapidity of spread from brain to the periphery—centrifugal spread—within a few days post inoculation, was likely due to the high viral dose inoculated directly into the brain. The extra-encephalic detection of lyssavirus antigen nearly exclusively in neurons indicates centrifugal spread via peripheral nerves, and fits with the accepted pathogenesis of lyssavirus infections , althougAlthough no marked lesions were present in the brain at the time of death, neuronal dysfunction was apparent from the neurological disease observed in our virus-inoculated bats. Although neurologic signs have not been previously described for LBV-infected bats, they correspond to those described previously for bats infected with other lyssaviruses , 64–66.The apparent consistency of the clinical presentation seen within each infection group, and the differences among the bat groups, suggests a link with the virus strain used. Specifically, all three bats inoculated with the Senegal strain developed marked sensitivity to external stimuli, and all three bats inoculated with the Ghana strain developed aggression and excessive vocalization. Interestingly, the Ghana LBV was reported to have been isolated from an apparently healthy straw-colored fruit bat in the frame of a non-targeted virus isolation study in a large colony comprising ca. 350,000 animals in Kumasi, Ghana . Here, aAll virus-infected, but no mock-inoculated, bats had a lymphocytic meningoencephalitis at death. This is similar to the histologic lesions seen in mice intracranially inoculated with lyssaviruses of different species . The preIn our study, several neurons in ganglia and other cells outside the central nervous system were lyssavirus antigen-positive. We used this information to deduce via which nerves the different LBV strains travelled from site of intracranial inoculation to extra-encephalic tissues . The GhaFor a lyssavirus to be suitable for use in pathogenesis studies, and specifically for causing infection in bats that leads to excretion of virus in the oral cavity, the virus needs to have the capacity to get to the excretion site. In straw-colored fruit bats that were naturally infected with LBV, salivary gland epithelial cells and tongue epithelial cells were shown to contain antigen . These tAlthough virus was found in surface epithelial cells of the tongue of two bats (bats 12 and 13), virus was not detected in any of the oral swabs. There are several possible explanations for this. First, immunohistochemistry to detect virus antigen in oral tissues may be more sensitive than hn-RT-PCR to detect viral RNA in oral swabs. Second, although there was virus in tongue surface epithelium, there may have been no excretion into the oral cavity. This does not fit well with what is known of the pathogenesis of other lyssaviral infections. Third, it may be that excretion was intermittent and swabs were taken at a time when virus was not excreted. Intermittent excretion has been proposed to explain alternating positive and negative results of serially collected oral swabs in other experimental lyssavirus infections in bats . HoweverIn conclusion, our study showed that intracerebral inoculation of each of three LBV strains into straw-colored fruit bats caused widespread infection of the brain, associated with meningoencephalitis, clinical signs consistent with rabies, progressing quickly to death of all virus-inoculated bats. Although group sizes were small, different clinical signs were observed and these were associated with the virus strains used. The distribution of virus antigen in extra-encephalic tissues indicated that the Ghana and Senegal LBV strains were able to utilize both sensory and motor neurons for dissemination to the periphery, and to infect epithelial cells of the tongue as a possible site of excretion. The Nigeria strain was rarely detected in any tissue outside the brain and may have lost the capability to use certain transport mechanisms in neurons, possibly due to multiple passages in cell culture. Based on the above results, both the Senegal and Ghana strains are suitable for further pathogenesis studies of LBV in the straw-colored fruit bat, a natural host of the virus. Now that we have shown that these strains are suitable, our next step will be to use a peripheral inoculation site, and thus a more natural route of transmission, for further pathogenesis studies in the straw-colored fruit bat.S1 Table(XLSX)Click here for additional data file.


Dengue fever is a mosquito-borne viral pandemic disease that is widespread in the tropical and subtropical areas. Dengue virus uses human mannose-binding receptor (MR) and DC-SIGN on macrophages as primary receptors, and CLEC5A as signaling receptor to sense the dengue virus invasion and then to signal and stimulate macrophages to secrete cytokines. But the interplay between MR/DC-SIGN and CLEC5A is unknown. Here we demonstrate a plausible mechanism for the interaction, i.e. MR/DC-SIGN first attracts the virus with high avidity, and the virus concurrently interacts with CLEC5A in close proximity to form a multivalent hetero-complex and facilitate CLEC5A-mediated signal transduction. Our study suggests that the cooperation between a high-avidity lectin-virus interaction and a nearby low-avidity signaling receptor provides a necessary connection between binding and signaling. Understanding this mechanism may lead to the development of a new antiviral strategy. Dengue fever is a mosquito-borne viral pandemic disease that is widespread in the tropical and subtropical areas, putting more than 4 billion people at risk, with 390 million infections and 96 million cases of illness annually, 2. UndeDengue infection starts with the attachment of the virus to the host cell surface, and then the virus diffuses along the cell surface to search for receptors on the host cell . The hosKD) in the micromolar range. As MR or DC-SIGN binds the virus with KD in the sub-nanomolar range, CLEC5A is unable to compete with MR and DC-SIGN in binding to the virus . But. But14].2/sec [It is known that after the dengue virus particle attaches to the cell surface and is stabilized, its diffusion constant at 37°C is less than 0. 025μm2/sec . ConsideKon value for CLEC5A binding to dengue virus due to the very weak association, the difference in dengue virus association between CLEC5A and MR/DC-SIGN is significant. Because of the high avidity between dengue virus and MR/DC-SIGN, it should not be possible for CLEC5A to bind to the virus independently, especially when MR and DC-SIGN are much larger molecules than CLEC5A, and would not favor prior binding of CLEC5A spatially. For these reasons, dengue virus may bind to MR/DC-SIGN first as the primary receptor, followed by a concurrent interaction with CLEC5A as the signal co-receptor in close proximity  of DC-SIGN has been shown to bind to specific envelope protein pairs on dengue virus, suggesting that DC-SIGN and dengue virus-CRD interaction is involved in a high order structure. In the In response to dengue virus infection, the GM-MDM cells use CLEC5A as an innate immune receptor, and this CLEC5A/DAP12 innate immune response is facilitated by the high avidity glycan binding receptors MR and DC-SIGN in proximity to form a hetero-multivalent complex. However, our data represent a steady state result, and the real dynamics of cell surface binding remain unclear. Though attempts to elucidate the entry pathway has been reported, a single-molecule imaging method may be required to understand the molecular mechanism of dengue virus infection.S1 Fig(a) Kinetic study of MR-Fc binding to dengue virus. (b) Kinetic study of Fc-DC-SIGN binding to dengue virus. (c) Fc-DC-SIGN and Fc-CLEC5A are sequentially immobilized on detection probe as 0.5nm/0.5nm ratio, and kinetic study was performed with dengue virus binding. (d) Fc-DC-SIGN and Fc-CLEC5A are sequentially immobilized on detection probe as 0.5nm/2.0nm ratio, and kinetic study was performed with dengue virus binding.Bio-layer interferometry  is used for the kinetic study of human receptor-Fc conjugates interacting with dengue virus. The biosensor (ForteBio) is immobilized with the receptor and reacts with dengue virus in Tyrode's solution at room temperature. The concentration of dengue virus in the study was determined based on the equivalent of dengue virus envelope protein. Data were analyzed with 1:1 binding as there is no other proper binding model to be fitted with this type of multivalent binding. (TIF)Click here for additional data file.S2 FigGM-MDM w/o IL-4 stimulation was generated as described and subsequently analyzed by flow cytometry for surface receptor expression and normalized with isotype control. Bars represent the SD of at least 3 independent experiments. One donor is shown.(TIF)Click here for additional data file.S3 Fig(a) Confocal fluorescence micrographs of human GM-MDM cells with indicated staining. (b) Confocal fluorescence micrographs of human GM-MDM cells + IL-4 stimulation with indicated staining.Staining of either dengue virus, or DC-SIGN, or MR, or CLEC5A as indicated. Images showing the colocalized pixels of DC-SIGN with MR, and CLEC5A with MR, and CLEC5A with DC-SIGN are also presented in false color. The dengue virus positive staining is gated to differentiate dengue virus presence and absence areas in dengue virus infected cells. (TIF)Click here for additional data file.S4 FigWe use MR/DC-SIGN as centre point, define specific radius for circle area: 25nm radius circle represents a single virus projection area, 50nm radius circle represents minimal area necessary for the whole embedded virus particle, 100nm radius circle represents 4- virus projection area, and 200nm radius circle represents multi-virus cluster projection areas.(TIF)Click here for additional data file.S5 Fig(a) The monovalent binding model cannot explain the function through the cooperation between binding receptor DC-SIGN and signaling receptor CLEC5A. (b) The multivalent binding model provides a better fit of current observation of functional and binding cooperation.(TIF)Click here for additional data file.


The data presented in this study were obtained from a novel approach to estimate a comprehensive loan acquisition cost. The latter includes commute costs and wage losses in addition to the monthly installment payments. These cost estimates represent the monetary value (in U.S. dollars) of the costs of driving to and from the installment lender storefront and that of the potential hourly wage losses, that is, wage loss from the driving time and the time spent at the loan office filling out the required paperwork to obtain the loan. Borrowers only get the net loan proceeds, that is, the original loan amount minus the comprehensive loan acquisition costs. The study area has 160 counties. It was created from the ESRI ArcGIS Map (a mapping software) using the spatial data from the U.S. Census, Topologically Integrated Geographic Encoding and Referencing (TIGER) Cartographic boundary files representing the geographies of states and counties. Using the U.S. road networks, the origin of the trip is a county seat in Arkansas and the destination of the trip is a county seat in a surrounding state of Tennessee, Mississippi, Louisiana, Texas, Oklahoma, and Missouri. The transportation networks were established using Google Earth/Directions to efficiently measure the travel time (distance). The average cost of a trip of 17 cents (U.S. dollar) was calculated based on the U.S. Department of Transportation Survey data, which identify important attributes of a typical vehicle used in a county such as model make, age of the vehicle, fuel consumption, etc. There are 10 occupational industry sectors where a typical borrower has a job. To estimate wage loss, the data were gathered from the U.S. Department of Labor, Bureau of Labor Statistics, namely, the Occupational Employment Statistics. Putting the missing pieces together, the data contain in this study improve our understanding of extra costs borne by borrowers located in the “loan desert” area. As expected, interior counties post high loan acquisition costs compared with border counties. The data from this study are useful to the public, businesses, policymakers, and researchers working on consumer finance. Specifications TableValue of the data●It is the first attempt in the literature to estimate a comprehensive loan acquisition cost, which includes commute costs and wage losses in addition to the monthly installment payments.●Cost estimates are presented based on the occupational industry sector where a typical borrower has a job.●It emphasizes the notion that the interest rate cap is unprofitable for businesses and imposes extra costs to borrowers as evidenced by “loan desert” in the interior of Arkansas.●It is useful to the general public, businesses, policymakers, and researchers working on consumer finance.1The detailed dataset is reported in the “Loan Acquisition Costs” MS Excel file, which summarizes the average loan acquisition costs for 10 occupational industry sectors  where a typical borrower has a job. Wages associated with each occupational industry sector are presented in the “Wages” MS Excel file. One of the requirements for loan application packet is that a representative borrower shall present a recent pay stub.t-statistic evaluated at the conventional levels of significance of 1%, 5%, and 10%. Results suggest very strong evidence of the difference of loan acquisition costs between border and interior counties. In addition, The average loan acquisition costs are presented based on the relative location of counties with respect to Arkansas borders, that is, border and interior counties. 22.1In the early 1900s, a new loan source emerged through the collaboration of lenders and consumer advocates. Their recommendations were captured in the Uniform Small Loan Law (USLL) of 1916. By 1940, all but nine states had adopted some version of this model legislation regulating cash installment loans.The Arkansas legislature chose to keep the constitutionally imposed rate cap of ten percent. In November 2010, however, amendment 89 to the Arkansas constitution passed with about 64% of the votes cast supporting it. This amendment raised the maximum interest rate for all loans, including cash installment loans, from 10% to 17%.An Annual Percentage Rate  of 17% has not induced small dollar cash installment lenders to operate in Arkansas. Revenues on loans with this cap do not cover loan production and servicing costs. Each of the six states  that border Arkansas have no rate cap, or a rate cap much higher than Arkansas. As a result, cash installment loans are offered by finance companies in each of these six states.A cash installment loan 1) has equal payments that fully amortize the debt after the borrower makes the last payment, and 2) has payments consisting of interest and an amount that reduces the principal owed. In these two ways, cash installment loans are like the familiar installment loans made to finance the purchase of appliances, furniture, or vehicles. Unlike these sales finance products, borrowers can use the proceeds from a cash installment loan in any manner they wish.Previous spatial analysis studies on small dollar loans focus on payday loans, which are short-term, lump-sum loans. Studies examine the locations picked by payday lenders By contrast, we examine small dollar cash installment loans. The specific contribution of this study is to provide a novel way to estimate the total acquisition costs of a cash installment loan by including (i) the commuting costs of driving to and from the installment lender; and (ii) the potential hourly wage losses for the driving time and the time spent at the loan office, filling out the required paperwork to obtain the loan. Borrowers only get the net loan proceeds, that is, the original loan amount minus the loan acquisition costs.2.2For convenience, we assume that the origin of each round trip is an Arkansas county seat and the destination is a county seat in another state. The standard practice in the spatial analysis is to use a built-in tool in the ArcGIS Map software, the Euclidean distance, to measure the distance between the origin and the destination. This standard practice fails to account for different landscapes, hills, valleys, and other natural barriers such as bodies of water. To address these limitations, we use the travel time in minutes and distance in miles from the Google Earth/Get Directions application.Then, we build a table of origin (county seats in Arkansas) and destination (county seats in two nearest counties of the bordering states). We employ a least cost approach using the minimum travel time between each origin/destination pair. We obtain a vehicle operating cost of $0.17, as detailed in LACij) from county-seat i in Arkansas to the closest county-seat j in any of the six surrounding states measures the overall costs of a loan is the sum of the commute cost and the foregone wages for the loan borrower, by industry classification. That is:TCCij represents the total commute costs expressed in dollars for the minimum travel time; VTi is the value of time for a consumer of county-seat i captured by the average hourly wage in industry sector, and tij is the minimum travel time between two county seats located in Arkansas and in any county seats of surrounding states; and, VPWTi equals one hour of foregone wages from completing the paperwork at the installment loan storefront.The proposed installment loan acquisition costs  is evaluated at 1%, 5%, and 10% significance levels, respectively.Finally, we test the hypothesis of no-difference between the average loan acquisition costs ($) in border and perimeter counties using a formal mean-comparison 2.3R package was employed to create A thematic ArcGIS map see  portrayst-statistics evaluated at 1% significance level. Data suggest high loan acquisition costs in interior counties (in blue) compared to border counties (in green) with low loan acquisition costs.y-axis shows the top and bottom fifteen counties based on the loan acquisition costs and the x-axis shows both the occupational industry sector and the loan acquisition costs expressed in U.S. dollars.2.4Data are consistent across occupational industry sectors. The average amount borrowed by Arkansas residents is about $1051 We propose and implement a novel way to estimate the total acquisition cost for Arkansas borrowers traveling out of state to obtain a cash installment loan. Overall, data show that it is expensive for Arkansans who reside in the interior counties to acquire small dollar installment loans. We study this expense across different occupational industry sectors. The data lend support to the notion that a small dollar cash installment loan desert exists in the interior counties of Arkansas. The complete table of loan acquisition costs data is provided in the MS Excel “Loan Acquisition Costs.xls” file along with wages data and the loan acquisition cost means comparison results.The authors received no financial support for the research, authorship, and/or publication of this article.