In cell culture, BXA at four nanomolar concentration achieved a 1.5–2.8 log reduction in virus titers of A(H7N9), including the NA-R292K mutant virus and highly pathogenic avian influenza viruses, whereas NA inhibitors or favipiravir required approximately 20-fold or higher concentrations to achieve the same levels of reduction. A(H7N9)-specific amino acid polymorphism at position 37, implicated in BXA binding to the PA endonuclease domain, did not impact on BXA susceptibility. In mice, oral administration of BXM at 5 and 50 mg/kg twice a day for 5 days completely protected from a lethal A/Anhui/1/2013 (H7N9) challenge, and reduced virus titers more than 2–3 log in the lungs. Furthermore, the potent therapeutic effects of BXM in mice were still observed when a higher virus dose was administered or treatment was delayed up to 48 hours post infection. These findings support further investigation of BXM for A(H7N9) treatment in humans.Human infections with avian-origin influenza A(H7N9) virus represent a serious threat to global health; however, treatment options are limited. Here, we show the inhibitory effects of baloxavir acid (BXA) and its prodrug baloxavir marboxil (BXM), a first-in-class cap-dependent endonuclease inhibitor, against A(H7N9),  The avian influenza A(H7N9) virus is an urgent concern for global public health due to the potential for pandemic spread2. In March 2013, the first outbreak in humans with an Asian lineage A(H7N9) were reported in China3. A(H7N9) was initially characterized as a low pathogenic avian influenza virus which caused mild disease in avian hosts4, however, of particular concern, were the acquisition of certain mutations (Leu226 in HA and Glu627 in PB2) which facilitated a switch to human-type receptor specificity enabling replication at temperatures present in the human respiratory tract7. Since early 2013, influenza A(H7N9) viruses have caused five distinct epidemic waves8, and laboratory-confirmed human cases admitted to hospital with A(H7N9) exhibited high case fatality rates9. The highest number of human cases with A(H7N9) occurred in China from October 2016 and the number of cases exceeded those reported in the previous waves11. Importantly, highly pathogenic A(H7N9) viruses were isolated from humans during the fifth epidemic wave10. As of October 2018, a total of 1,567 laboratory-confirmed human cases (with 615 deaths) of A(H7N9) infections in humans have been reported12.Influenza pandemics arise when novel reassortant influenza A viruses acquire zoonotic potential and adapt to enable efficient and widespread human-to-human infection13. While the A(H7N9) viruses are resistant to M2-ion channel blockers, treatment with oseltamivir and peramivir have been reported to be effective in patients with A(H7N9) infection14, with oseltamivir the most widely used drug15. Importantly, evidence for the therapeutic effects of oseltamivir treatment has been accumulating; however, the available NAIs show limited ability to reduce viral titers, therefore, there may be limited virologic or therapeutic advantages, especially in patients with severe influenza with high viral loads18. In addition, the emergence of drug resistant mutants have been detected after NAI treatment against seasonal and zoonotic influenza A virus infections23. In particular, the arginine to lysine mutation at position 292 (N2 numbering) in the NA (NA-R292K) confers a reduced susceptibility to all approved NAIs, and has been identified in A(H7N9) clinical isolates, such as A/Shanghai/1/2013 and A/Taiwan/1/201325. Therefore, novel antivirals that overcome the current limitations for treatment of A(H7N9) infection are urgently required to decrease both morbidity and mortality in infected subjects and to assist in the preparedness to help mitigate widespread transmission in possible future pandemics.Antiviral medications are of critical importance to reduce the burden of disease caused by A(H7N9) and the U.S. Centers for Disease Control and Prevention (CDC) recommends the use of antivirals for treatment of all hospitalized cases of human infection with novel influenza A viruses associated with severe disease26 and thereby plays a crucial role during both the replication and transcription stages of the viral life cycle. As a result the cap-snatching mechanism is considered a promising antiviral target27. The cap-dependent endonuclease (CEN) in the PA subunit cleaves bound-capped RNAs 10–13 nucleotides from the 5′ ends of nascent transcripts to generate a primer for the synthesis of viral mRNA. The active center of CEN is also highly conserved across seasonal, pandemic, and highly pathogenic avian influenza viruses28, indicating that CEN inhibitors have the potential to be broadly-active anti-influenza drugs30.The influenza RNA polymerase complex, composed of the PA, PB1 and PB2 subunits, helps mediates a unique “cap-snatching” mechanism33. To enhance oral absorption of BXA, the phenolic hydroxyl group was modified, yielding the prodrug BXM. Notably, BXA exhibits broad antiviral activities against several subtypes of influenza A and B viruses in vitro, and co-crystal structures of PA endonuclease domain from influenza A and B viruses support broad spectrum activity for this class of compounds35. Therefore, BXM is anticipated to be an additional and alternative option for treatment of avian-origin influenza A viruses; however, information on the in vitro potency of BXA and in vivo efficacy of BXM is still limited. In the present study, we report the antiviral activities of BXA and BXM against human influenza A(H7N9) viruses, including highly pathogenic avian influenza viruses. Our findings support further investigation of the therapeutic efficacy of BXM treatment in A(H7N9)-infected patients.Baloxavir marboxil (BXM), which is converted metabolically to its active form baloxavir acid (BXA), is an orally available CEN inhibitor that has recently been approved for clinical use (single dose of 40 mg for patients 40 kg to <80 kg or 80 mg for patients ≥ 80 kg) in adults and adolescents in Japan and the United States37, which is involved in BXA binding in the endonuclease domain (Table 38. To examine whether BXA possessed inhibitory activity against human A(H7N9) virus in vitro, we selected strains from subtypes A(H7N9) and A (H7N3), including highly pathogenic avian influenza viruses, isolated from 2013 to 2018 harboring alanine or serine at residue 37 in the PA. To compare the degree of inhibition of virus replication by approved drugs, a yield reduction assay using Madin-Darby canine kidney (MDCK) cells was employed. The mean concentration achieving 90% (1-log) reduction in virus titer (EC90) values of approved drugs and virus titers under different drug concentrations are shown in Table 35 and exhibited comparable potency against A/Anhui/1/2013 (H7N9) harboring the NA-R292K substitution to the wild-type, indicating no-cross resistance with NAIs was observed. BXA also exhibited comparable potency against H7 low and highly pathogenic avian influenza viruses to A/Anhui/1/2013 (H7N9) strain. Notably, BXA at four nanomolar concentration achieved a 1.5–2.8 log reduction in viral titers (Supplementary Table in vitro.Avian-origin influenza A(H7N9) viruses typically harbor a polymorphic alanine to serine substitution at residue 37 in the PA (A37S)in Table 32,38. To50) of A/Anhui/1/2013 (H7N9). All vehicle-treated mice died within 7 days post-infection (dpi) and mean day to death was 6 days  in a lethal infection model, mice were inoculated with 10.4 times of 50% mouse lethal dose 33. These results suggest that BXM has profound inhibitory effects on viral replication in the lungs of mice infected with A(H7N9).In order to examine the inhibitory effects of BXM on the viral replication of A/Anhui/1/2013 (H7N9) 42. Furthermore, A(H7N9) infection-associated inflammation in the lungs resulted in a rapidly progressive pneumonia and development of acute respiratory distress syndrome in the majority of hospitalized patients43. Therefore, the impact of BXM treatment upon proinflammatory cytokines and chemokines production in the lung of mice inoculated with A/Anhui/1/2013 (H7N9) was evaluated. Treatment of the A(H7N9)-infected mice with BXM at 5 and 50 mg/kg twice a day for 1 and 5 days resulted in a significantly less pronounced production of both proinflammatory cytokines and chemokines when compared with vehicle-received mice, consistent with the reduction of virus titers in the lungs of mice -6, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and interferon (IFN)-γ, is markedly elevated in A(H7N9)-infected patients44, and therefore we next investigated the protective effect of BXM in a high-dose challenge model of A(H7N9) infection. Mice were inoculated with 31.1 MLD50 of A/Anhui/1/2013 (H7N9) and were then treated with OSP or BXM beginning immediately after virus inoculation. The mice administered with OSP at 5 and 50 mg/kg twice a day for 5 days died within 6 and 8 dpi, respectively, and no significant difference was observed on survival time up to 21 dpi compared to vehicle-treated group  challenge in mice increased in a dose-dependent manneroup Fig. . Strikinoup Fig. . Moreoveoup Fig. , consist50 of A/Anhui/1/2013 (H7N9) were treated with BXM starting after 24 and 48 hours after infection, and subsequently survival and body weight loss was monitored. Mice without treatment died 5 to 7 days after infection, consistent with findings presented in Fig. To further investigate the therapeutic effect of BXM, mice infected with 10.4 times of MLDin vitro antiviral activity of BXA against influenza A(H7N9) viruses, including highly pathogenic avian influenza viruses, and the therapeutic effect of BXM in vivo in murine lethal models employing influenza A(H7N9) virus. In the yield reduction assays in vitro, BXA showed superior potency against replication of A/Anhui/1/2013 (H7N9) and recent isolates of highly pathogenic avian H7 viruses compared to the approved drugs. Given that the A/duck/Japan/AQ-HE28-3/2016 (H7N9) and A/duck/Japan/AQ-HE29-22/2017 (H7N9) strains based on PA gene segments are closely related phylogenetically to human A(H7N9) isolated in the fifth wave . Furthermore, BXM protected from lethal infection and exhibited significant decreases in both virus replication and proinflammatory responses in mice infected with A/Anhui/1/2013 (H7N9). Further investigations, to study the inhibitory activity of BXA against recent isolates of A(H7N9), are warranted to examine further the utility of BXM.In this study, we evaluated the 46 or NAI-resistant viruses have emerged during treatment48. In this study, A/Anhui/1/2013 (H7N9) bearing the R292K substitution in the NA protein conferred resistance to oseltamivir acid  were detected following BXM treatment in mice infected with A(H7N9). Given that A(H7N9) harbors the polymorphic A37S substitution adjacent to Ile38, there is a hypothetical possibility that A37S effects on the frequency of the detection of Ile38 substitutions. Hence, it would be worth undertaking resistance isolation experiments with A(H7N9) virus and analyzing the genetic compatibility of the viruses bearing both A37S and I38T substitutions.Although NAI-treatment for A(H7N9) infection is effective and recommended, it was reported that NAI-resistant variants e.g. NA-R292K) were isolated from human cases92K were 5. Comparable results were obtained in this study. In our model, we showed that only 1-day dosing of BXM at 0.5 mg/kg twice a day were sufficient for significant reduction of virus titers in the lungs of mice. Furthermore, 5 or 50 mg/kg twice a day dosing of BXM achieved dramatic reductions in virus titers compared to the vehicle and OSP-treated groups and resulted in significant improvements in survival. Five-day dosing of BXM at 5 or 50 mg/kg twice a day also achieved dramatic reductions in virus titers and extended the treatment window, which indicated that repeating dose of BXM might be a reasonable option in severe cases of A(H7N9) infection. It has been suggested that the target plasma BXA concentration 24 h after a single-dose (C24) be set at 6.85 ng/mL in non-clinical and clinical studies51. The plasma concentration of BXA could be maintained above the target concentration of 6.85 ng/mL for at least 5 days following oral administration of BXM at 40 mg in humans51. Although it is difficult to set a clinically equivalent dose regimen in mice owing to the crucial difference in half-life of BXA in plasma after oral BXM administration between humans (85.9 hours at 40 mg BXM) and mice (2.24 to 3.14 hours at 0.5 to 50 mg/kg BXM), the C24 of BXA after 1-day dosing of BXM at 50 mg/kg twice a day, as well as C120 of BXA after 5-day dosing of BXM at 5 mg/kg twice a day in mice are expected to be lower than or close to those in humans51. Therefore, the dosages used in the mouse model of BXM at 50 mg/kg twice a day for 1 day or at 5 mg/kg twice a day for 5 days are comparable to the clinical dosages. On the other hand, a C120 of BXA after 5-day dosing of BXM at 50 mg/kg twice a day may be higher than that in humans. The existing evidence strongly suggests that BXM is has superior protective efficacy against A(H7N9) infection in murine models when compared to OSP, although further pharmacokinetic and pharmacodynamic analyses in mice and humans are required for a more complete understanding of these increased therapeutic effects exerted by BXM.The therapeutic effect of oral BXM administration was investigated following A(H7N9) challenge in a lethal infection murine model. In a previous report, 5 days dosing of OSP had limited effect on the inhibition of body weight loss and the concomitant reduction of virus titers in mice infected with A/Anhui/1/2013 (H7N9) strain52. It has been shown that the potency of oseltamivir acid was diminished at higher multiplicities of infection (MOI) in MDCK cells in an MOI-dependent manner53, and OSP exhibited lower efficacy against higher infectious doses in a ferret model of influenza A(H5N1) virus infection54. Consistent with these observations, we confirmed that OSP did not reduce either morbidity or mortality in mice infected with high infectious doses of A(H7N9). We can infer from these findings that the therapeutic efficacy of OSP against influenza A virus infection attenuates dependent on the infectious dose. It is therefore particularly noteworthy that we demonstrated that 1-day dosing of BXM at 5 or 50 mg/kg twice a day showed greater efficacy against high dose A(H7N9) infection when compared to NAI OSP. Notably, favipiravir, which targets the viral RNA-dependent RNA polymerase, also exerted a strong effect upon the reduction of virus titers in the lungs of mice infected with high titers A(H7N9) when compared to NAIs5. These results suggest that inhibition of the viral RNA polymerase machinery, particularly the “cap-snatching” mechanism, may have the potential to be more effective for reducing virus titers and provide improved benefits for the treatment of severe influenza infections with high viral burdens.Tsang and co-workers reported that patients with high viral load correlated with the severity of influenza symptoms56. In addition, high levels of proinflammatory cytokines/chemokines were produced in the lungs of mice and cynomolgus macaques infected with A(H7N9) clinical isolates that replicated efficiently in the lungs57. In a murine model, suppression of proinflammatory cytokine/chemokine production positively correlated with both a reduction of virus titers in lungs and the disease severity58. Proinflammatory cytokines and chemokines are highly induced in the early phase of influenza virus infection and are associated with airway inflammation59; thus, BXM may serve to ameliorate severe influenza pneumonia due to exerting an inhibitory effect upon the production of IL-6, MCP-1 and MIP-1α in the early stages of virus infection. Here we have shown that IFN-γ production in the lungs was also suppressed by treatment with BXM. IFN-γ production in macrophages during influenza virus infection is thought to be critically important in sustaining the cytokine/chemokine storm, and IFN-γ acts to upregulate CXCL10 transcription in airway epithelial cells, which result in infiltration of effector T cells to the lung airways61. Reduction of IFN-γ thus likely limits cytokine overproduction in the lungs. These results suggest that BXM prevented virus replication followed by a concomitant decrease in the production of proinflammatory cytokines and chemokines. Thus, inhibition of virus replication in the early stages of infection appears therefore to be critically important for the amelioration of host dysfunction which likely serves to mediate the therapeutic effects.In human infection with A(H7N9), virus-induced proinflammatory cytokine and chemokine dysregulation in the lungs or serum contributes to disease severityin vitro and in vivo compared to other currently approved antivirals. Although further investigations are required to clarify therapeutic effects against highly pathogenic avian-origin influenza A viruses in vivo, the existing evidence supports and warrants the consideration of BXM as an alternative therapeutic option for the treatment of A(H7N9) infection in humans.In conclusion, this study demonstrates the high potency of BXA and BXM against influenza A(H7N9) viruses Baloxavir marboxil (BXM) and baloxavir acid (BXA) were synthesized at Shionogi & Co., Ltd.. Oseltamivir acid, laninamivir were purchased from Toronto Research Chemicals Inc. . Oseltamivir phosphate and zanamivir hydrate were obtained from Sequoia Research Products Ltd. . Favipiravir was supplied by PharmaBlock Sciences, Inc. .2 in minimum essential medium  supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/L L-glutamine, 50 units/mL penicillin, 50 µg/mL streptomycin and 0.05% sodium hydrogen carbonate. The non-mouse adapted influenza A/Anhui/1/2013 (H7N9) virus, which is a clinical isolate and is pathogenic in mice41, A/duck/Japan/AQ-HE28-3/2016 (H7N9), A/duck/Japan/AQ-HE29-22/2017 (H7N9) and A/duck/Japan/AQ-HE30-1/2018 (H7N3) were propagated in embryonated chicken eggs and harvested from virus-containing allantoic fluids. A/Anhui/1/2013 (H7N9) and A/duck/Japan/AQ-HE28-3/2016 (H7N9) possess low pathogenicity, A/duck/Japan/AQ-HE29-22/2017 (H7N9) and A/duck/Japan/AQ-HE30-1/2018 (H7N3) possess high pathogenicity45. Recombinant A/Anhui/1/2013 (H7N9) virus harboring NA-R292K was generated by plasmid-based reverse genetics62. Recombinant virus was propagated in embryonated chicken eggs and harvested from virus-containing allantoic fluids. Infectious titers were determined by standard 50% tissue culture infectious dose (TCID50) assay in MDCK cells.The Madin-Darby canine kidney  cells were maintained at 37 °C under 5% CO50/well. The infected cells were incubated at 35 °C under 5% CO2 for 1 hour and wash out the virus inoculum, followed by addition of the fresh medium including 2.5 µg/ml trypsin and defined concentrations of test compounds. BXA and favipiravir were dissolved in dimethyl sulfoxide and NAIs were dissolved in distilled water. The cells were incubated at 35 °C under 5% CO2 for 24 hours and virus titers (TCID50/mL) in the culture supernatants were determined in MDCK cells. The 90% effective concentration (EC90) was calculated as the concentration decreasing the virus titers in the culture supernatant to 10% of untreated control values by the linear interpolation method.Two days prior to infection, MDCK cells were seeded in 96-well plates and the cells were infected with each virus at 100 TCID63 with default parameters. Sequence data of PA genes obtained in the present study were compared with those of other reference strains presented in a previous report45.PA sequences of 10,312 clinical isolates for A(H1N1), 13,185 for A(H3N2), 196 for A(H5N1) and 1,094 for A(H7N9), were downloaded from the National Center for Biotechnology Information (NCBI) and Global Initiative on Sharing All Influenza Data (GISAID) on October 24, 2018. Amino acid sequences of the PA protein were aligned by the ClustalW program in the component of Pipeline Pilot 2018 (BIOVIA), and conservation of amino acid residues in close proximity to the BXA was calculated within individual influenza virus subtypes. The nucleotide sequences were phylogenetically analyzed based on PA genes of H7 avian influenza viruses by the maximum-likelihood method with a Tamura–Nei model and bootstrap analysis (n = 1000) using MEGA 7.0 software5 TCID50  or 1.2 × 106 TCID50  of A/Anhui/1/2013 (H7N9) virus. On the morning of the first day, the first dosing was administered within a few seconds after virus inoculation under anesthesia in the morning of the first day (defined as immediately after virus inoculation). Mice were treated with BXM  twice a day  for 1 or 5 day(s) by oral gavage. BXM was suspended with 0.5 w/v% methylcellulose (MC). For the controls, vehicle (0.5 w/v% MC) or oseltamivir phosphate  was administrated twice a day for 5 days by oral gavage. OSP was dissolved with 0.5 w/v% MC. Dosing volume was 10 mL/kg calculated by body weight before each dosing. Survival rates and body weight changes were then monitored through a 21 (high dose infection model) or 28-day (low dose infection model) period after the infection (n = 10/group). Virus titers in the lungs of mice at indicated time points were determined in MDCK cells. The viral RNAs derived from lung homogenates of BXM-treated mice was extracted by PureLink Viral RNA/DNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Reverse transcription reaction, amplification of cDNA and sequencing reaction were performed as previously reported64. Primers used in this study were as follows; PA-1F, 5′-ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3′ and PA-955R, 5′-TGCATTTGATTGCATCATATAG-3′. Sequence analysis of PA N-terminal domain (the PA gene of A/Anhui/1/2013 [H7N9] strain) was performed by Sanger sequencing method using the 3500/3500xL genetic analyzer (Life Technologies). All animals were housed in self-contained units (Tokiwa Kagaku) at the BSL-3 and ABSL-3 facilities of the Faculty of Veterinary Medicine, Hokkaido University, Japan. Animal experiments were performed according to the guidelines of the institutional animal care and use committee of Hokkaido University . The mice were euthanized when they lost greater than 30% of their body weight compared with their pre-infection weight.Six-week-old female BALB/c mice  were maintained under a controlled temperature environment and humidity. Under anesthesia , mice were infected intranasally (50 µL/mouse) with 4.0 × 10Under anesthesia, mice were infected intranasally with low dose of A/Anhui/1/2013 (H7N9) virus. Mice were treated with BXM (5 or 50 mg/kg/dose) twice a day for 5 days by oral gavage beginning immediately, 24 or 48 hours after virus inoculation. For the controls, vehicle or OSP (5 mg/kg/dose) was administrated twice a day for 5 days by oral gavage beginning immediately after virus inoculation. Dosing volume was 10 mL/kg calculated by body weight before each dosing. Survival rates and body weight changes were then monitored through a 21-day period after the infection (n = 5/group).5 TCID50 of A/Anhui/1/2013 (H7N9) strain at 1 (only for 1-day treatment group), 3 and 5 dpi. The collected lungs were homogenized, and each sample was processed according to the manufacturer’s protocol.In experiment 1, levels of proinflammatory cytokines and chemokines, including interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α and interferon (IFN)-γ in the lungs, were quantitatively determined using Quantikine ELISA (R&D Systems). The lungs were collected from the virus titer experiments infected with 4.0 × 10P values < 0.05 were considered statistically significant.For the comparison of the survival time after infection between each BXM-treated group and vehicle-treated or OSP-treated group, the log-rank test was applied in experiment 1 and 2. For the comparison of body weight changes of mice throughout the treatment period, virus titers in the lung tissues or cytokines and chemokines levels in the lung tissues between each BXM-treated group and vehicle-treated or OSP-treated group at each time point were evaluated using Dunnett’s multiple-comparison method in experiment 1. Statistical analysis was performed using the statistical analysis software SAS version 9.2 for Windows . All animals were housed in self-contained units (Tokiwa Kagaku) at the BSL-3 and ABSL-3 facilities of the Faculty of Veterinary Medicine, Hokkaido University, Japan. Animal experiments were performed according to the guidelines of the institutional animal care and use committee of Hokkaido University . The mice were euthanized when they lost greater than 30% of their body weight compared with their pre-infection weight.Dataset 1


Y88F/H547A mutant attenuated Tat-induced inhibition of dopamine uptake observed in wild-type hDAT. Y88F/H547A displayed an attenuation of zinc-augmented [3H]WIN35,428 binding, increased basal dopamine efflux, and reduced amphetamine-induced dopamine efflux, indicating this mutant alters transporter conformational transitions. These findings further demonstrate that both tyrosine88 and histidine547 on hDAT play a key role in stabilizing basal dopamine transport and Tat-DAT integration. This study provides mechanistic insights into developing small molecules to block multiple sites in DAT for Tat binding.Dysregulation of dopaminergic system induced by HIV-1 Tat protein-mediated direct inhibition of the dopamine transporter (DAT) has been implicated as a mediating factor of HIV-1 associated neurocognitive disorders. We have reported that single point mutations on human DAT (hDAT) at tyrosine88 (Y88F), lysine92 (K92M), and histidine547 (H547A) differentially regulate basal dopamine uptake but diminish Tat-induced inhibition of dopamine uptake by changing dopamine transport process. This study evaluated the effects of double (Y88F/H547A) and triple (Y88F/K92M/H547A) mutations on basal dopamine uptake, Tat-induced inhibition of DAT function, and dynamic transport process. Compared to wild-type hDAT, the V The continuous exposure of the central nerves system to HIV-1 viral proteins, inflammation, and antiretroviral agents results in neuropathological and neurocognitive deficits observed in the patients with HAND7. Transactivator of transcription (Tat) protein, one of seven HIV-1 viral proteins, has been shown to play a critical role in HIV-1 viral replication as well as the development of HAND8, which can be exacerbated by concurrent cocaine abuse9. Thus, developing an intervention strategy in the early stage of HIV-1 infection would prevent the development of neurocognitive dysfunction in HIV-1 infected individuals.About thirty-seven million people are currently living with HIV-1 infection worldwide, leading to a significant global public health problem. While the effective antiretroviral therapies significantly reduced the mortality rate in the patients with HIV-1 infection, nearly 50% of HIV-1 infected patients have various degrees of neurological complications that are referred to as HIV-1-associated neurocognitive disorders (HAND)11, and motivation13. Dopamine (DA) transporter (DAT) is a presynaptic membrane protein that reuptakes the released DA from the synaptic cleft back into cytosol, maintaining a stable DA homeostasis. The DAT activity is directly inhibited by HIV-1 Tat protein and cocaine, which synergistically enhances synaptic DA levels9. The dysregulation of DA system is a mediating factor of HAND as well as a factor in cocaine abuse15. Using computational modeling and experimental approach, we have identified several key residues on human DAT (hDAT), which are crucial for Tat-hDAT interaction and dynamic transport process9. Furthermore, we have demonstrated that in vitro exposure to Tat reduced reuptake of DA via hDAT in cells18 and rat striatal synaptosomes19. The inhibitory effect of Tat on DAT function results from Tat directly interacting with DAT21. Single point mutations of hDAT at tyrosine88 , lysine 92 , histidine547  differentially alter basal DA uptake but attenuate the Tat inhibitory effects on DA transport18. For example, DA uptake is decreased in K92M and increased in H547A, respectively; however Y88F mutant preserves basal DA uptake18. Notably, the mutational effects on normal DA uptake and Tat inhibitory effect on DAT function are associated with alterations of transporter conformational transitions9.Normal dopaminergic transmission is important for maintaining different brain activities including attention, learning, memory22. Recent studies have demonstrated that novel SRI-compounds exhibit a partial antagonistic role in DAT function as allosteric modulators25. We have reported that SRI-30827, one of the novel allosteric modulators, blocks Tat interaction with DAT26. Thus, identifying the specific binding sites on hDAT for Tat and its role in DA transport process could be beneficial to attenuation of the inhibitory effect of Tat on DAT-mediated dopaminergic transmission. On the other hand, through an allosteric modulatory mechanism, the inhibitory effect of Tat on DAT function can also be diminished by targeting the specific DAT residues that are distinct from Tat binding sites. However, based on our computational structural models for Tat binding with hDAT, the interaction of Tat with hDAT involves multiple residues of DAT21 and our previous results obtained from single point mutations of DAT only present the role of a particular residue in Tat-DAT interaction. Therefore, this study investigated the mutational effects of Y88F/H547A and Y88F/K92M/H547A on basal DAT function, Tat inhibitory effect on DA uptake, and dynamic DA transport process.We have demonstrated that Tat protein inhibits DAT function in an allosteric modulatory mechanism20, hDAT residues Y88, K92, and H547 could form hydrogen bonds with residues K19, P18, and R49 of HIV-1 Tat, respectively. According to our previous computational and experimental results, single mutation of either hDAT Y88 (Y88F), K92 (K92M) or H547 (H547A) could significantly attenuate the binding between DAT and Tat18. As shown in Fig. Based on the constructed hDAT-Tat binding model in our previous workmax of [3H]DA uptake, respectively, while Y88F preserved normal DA uptake18. The current study determined the multiple mutations of these residues on DA uptake and DAT binding sites. As shown in Table 5 cells), the Vmax values were increased by 92% in Y88F/H547A  = 2.33, p < 0.05) and dramatically decreased in Y88F/H547A/K92M  = 16.2, p < 0.001), respectively. Additionally, compared to WT hDAT (1.21 ± 0.17 µM), the Km value in Y88F/H547A was increased by 250%  = 2.54, p < 0.05) but not altered in Y88F/K92F/H547A.We have demonstrated that single point mutations on hDAT at His547 (H547A) and Lys92 (K92M) displayed a 196% increase and a 71% decrease in the V3H]WIN35,428 binding sites share pharmacological identity with the DA uptake site and is also part of the cocaine binding domain28. Kinetic analysis of [3H]WIN35,428 binding was conducted to determine the effects of multiple mutations of hDAT on the DA binding site. As shown in Table max nor Kd values of [3H]WIN35,428 were altered in Y88F/H547A, whereas Y88F/K92M/H547A displayed a 61% decrease in the Bmax value (t(14) = 2.4, p < 0.05) and a 199% increase in the Kd value (t(14) = 4.4, p < 0.001). Thus, due to the significant reduction on DA uptake for Y88F/K92M/H547A, only the Y88F/H547A mutant was included for the rest of experiments.In the hDAT, the [3H] DA uptake in WT hDAT and Y88F/H547A (Table 50) for DA . In contrast, compared to WT hDAT, Y88F/ H547A displayed an increase in IC50 for cocaine  and GBR12909 . We also evaluated the mutational effect of Y88F/H547A on the IC50 of DA, cocaine, and GBR12909 for inhibiting [3H]WIN35,428 binding  and cocaine  = 3.73, p < 0.001]. No difference in IC50 GBR12909 inhibiting [3H]WIN35,428 binding was observed between WT hDAT and Y88F/H547A.To characterize the influence of Y88F/H547A on DA transport and the specific binding site in hDAT for DA, cocaine, GBR12909, and amphetamine (AMPH), we determined the ability of these DAT inhibitors or substrate to inhibit . No significant mutation × PMA treatment interaction  was found. Addition of PMA reduced the Vmax values in WT hDAT by 51%  and Y88F/H547A by 60% , respectively, compared to their respective vehicle controls. Similarly, two-way ANOVA analysis on the Km values revealed significant main effects of mutation  and PMA treatment . No significant interaction of mutation × PMA treatment was observed. The addition of 1 µM PMA reduced Km values by 53% in WT hDAT  and 63% in Y88F/H547A , respectively. These results suggest that the double mutation of Tyr88 and His547 attenuates the single mutant H547A-increased sensitivity of PMA-induced decrease in DA uptake.The activation of PKC by phorbol 12-myristate 13-acetate (PMA), a PKC activator, decreases DA uptake through DAT3H]DA uptake observed in WT hDAT18. Since the triple mutant (Y88F/K92M/H547A) displayed an extremely low Vmax of [3H]DA uptake, only the double mutant (Y88F/H547A) was tested for its effect on Tat-induced inhibition of DA uptake. Based on the computational modeling predictions . As shown in Fig. 1–86 (rTat1–86) significantly reduced the specific [3H] DA uptake in WT hDAT by 31% relative to its control . However, the Tat-induced reduction of [3H] DA uptake in WT hDAT was attenuated in Y88F/H547A compared to its control , suggesting that the double mutation of Tyr88 and His547 attenuates Tat’s inhibitory effect on DA uptake.Our previous studies demonstrated that single point mutation of Tyr88 (Y88F) or His547 (H547A) attenuated Tat-induced inhibition of DA uptake and [3H]WIN35,428 binding. Addition of Zn2+ is able to partially reverse an inward-facing state to an outward-facing state32. On the basis of this principle, the addition of Zn2+ to WT hDAT would inhibit DA uptake, whereas in a functional mutation of DAT, Zn2+ might diminish the preference for the inward-facing conformation and thus enhance DA uptake. As shown in Fig. 3H]DA uptake in WT hDAT and Y88F/H547A revealed significant main effects of mutation , zinc , and mutation × zinc interaction . The addition of zinc significantly decreased the [3H]DA uptake in WT hDAT and its mutant in a concentration dependent manner. Compared to the respective controls (in absence of Zn2+), the addition of Zn2+ (10 and 100 µM) produced a similar reduction of [3H]DA uptake in WT  but a greater reductions  in Y88F/H547A (ps < 0.01). This suggests that the double mutation of Y88/H547 does not affect zinc-induced regulation of DA uptake. In contrast, as shown in Fig. 3H]WIN35,428 binding revealed significant main effects of mutation , zinc , and mutation × zinc interaction . Compared to the respective controls (in absence of Zn2+), addition of Zn2+ (10 and 100 µM) significantly increased [3H] WIN35,428 binding in WT hDAT . The Zn2+ (10 and 100 µM)-induced increase in [3H]WIN 35,428 binding in WT hDAT was significantly diminished in Y88F/H547A , suggesting that Y88F/H547A attenuates zinc-induced increase in [3H]WIN35,428 of hDAT by altering transporter conformational transitions.Tat protein has been shown to regulate DA transport allosterically, which may lead to alteration of transporter conformational transitions3H]DA in PC12 cells expressing WT hDAT and Y88F/H547A  = 59.38, p < 0.0001], time , and mutation × time interaction . Post hoc analyses showed that compared to WT hDAT, DA efflux levels were significantly elevated at 1 and 10 min .To further determine the effect of Tyr88/His547 mutation on transporter conformational transitions, we examined basal efflux levels of [47A Fig. . twenty 34. AMPH-stimulated [3H] DA efflux in PC12 cells transfected with WT hDAT and its mutants was determined under a stable baseline of DA efflux  = 1.92, p = 0.14, one-way ANOVA). As shown in Fig. 3H]DA efflux in WT hDAT and mutants in a concentration dependent manner. Two-way ANOVA analysis on the Vmax values revealed significant main effects of mutation , AMPH  and mutation × AMPH interaction . As shown in Table max values of [3H]DA efflux in response to AMPH were reduced in H547A (0.22 ± 0.04) and Y88F/H547A (0.20 ± 0.03) compared to WT hDAT . No difference in Km was found among WT hDAT and all mutants.AMPH acts as a substrate for DAT which subsequently reverses the transporter leading to DA effluxlux Fig. , showing3H]DA uptake in WT and Y88F/H547A  and Y88F/H547A . Next, we performed the kinetic analysis of [3H]DA uptake in WT and Y88F/H547A in presence of AMPH . As shown in Fig. max of [3H]DA uptake reveled significant main effects of mutation  = 9.50, p < 0.01) and AMPH  = 11.99, p < 0.01). No significant interaction of mutation × AMPH treatment was found  = 1.61, p = 0.22). Post hoc analyses show that the Vmax value in Y88F/H547A (18.9 ± 2.81 pmol/min/105 cells) was higher than that in WT hDAT  = 2.49, p < 0.05, n = 5/group) in the absence of AMPH. AMPH decreased the Vmax in both WT hDAT  = 2.76, p < 0.01) and Y88F/H547A  = 2.58, p < 0.05) compared with their respective controls. There was a significant difference in the magnitude of the AMPH-induced decrease in Vmax values between WT hDAT and Y88F/H547A (t(8) = 1.86, p < 0.05). Furthermore, two-way ANOVA analysis on Km values revealed significant main effects of mutation  and AMPH ,whereas no significant interaction of mutation × AMPH treatment was found . In the absence of AMPH, the Km values of [3H]DA uptake was greater in Y88F/H547A (5.12 ± 0.76 µM) than that in WT hDAT , which was attenuated in Y88F/H547A (7.24 ± 1.15 µM).Double mutation of Tyr88 and His547 could induce DAT conformational change by altering AMPH interaction with DAT. We first examined the affinity for AMPH to inhibit WIN35,428 binding, increased basal DAT-mediated DA efflux, and reduced AMPH-stimulated DA efflux. Collectively, these findings suggest that targeting DAT residues Tyr88 and His547 may have potential therapeutic benefits on Tat-induced dysregulation of dopaminergic system seen in patients with HAND.Our recent studies demonstrated that single hDAT mutants, Y88F, K92M, and H547A differentially regulate DAT reuptake but attenuate Tat’s inhibitory effect on DAT function. This study sought to determine whether multiple mutations on these hDAT residues (Y88F/H547A) and (Y88F/K92M/H547A) influence the direct interactions between Tat and DAT. There were three major findings. First, compared with WT hDAT, Y88F/H547A enhances V35. This YYY motif has hydrophobic contact with a typical U-turn loop associated with extracellular loop 6 (EL6) and transmembrane 10 (TM10a). Any structural changes affecting the stability of the YYY motif could destabilize the R85-D476 salt bridge of hDAT, and the salt bridge is key for the conformational conversion39. H547 is expected to significantly affect the hDAT stability because H547 is adjacent to Y548 of the YYY motif35, which is consistent with the observed increase in DA uptake by the H547A mutation18. On the other hand, the side chain of K92 residue (TM1b) forms a favorable salt bridge with the side chain of D313 (TM6a) during the molecular dynamics simulation on the hDAT-DA binding complex, and the bridge is also essential for the conformational conversion of hDAT required for the DA uptake process40. For this reason, disrupting the K92-D313 salt bridge will likely impair or slow down the DA uptake process, which has been validated pharmacologically by mutations on K92 (K92M)17 and D313 (D313N)41. In addition, the aromatic ring on the side chain of Y88 is sandwiched by TM1b and extracellular loop 4 (EL4) in a hydrophobic region of hDAT. Therefore, mutation of Y88 to phenylalanine retains the normal reuptake function of hDAT and demonstrated no difference compared to WT hDAT17. However, Y88F increased the Km of DA binding by about 50%, suggesting that Y88F may slightly alter the local structure around Y8817. In the current study, the double mutant Y88F/H547A retains the increased Vmax observed in H547A, suggesting a critical role of the mutated H547 residue in regulating transporter activity. In contrast, the combination of the mutated residues Y88, K92, and H547 does not follow a simple math addition, because compared to WT hDAT, a 96% reduction of Vmax was observed in Y88F/K92M/H547A, whereas the Vmax was decreased by 71% in K92M17. These results suggest that the inclusion of the K92 mutation in the K92-D313 salt bridge is a critical factor in the differential DAT kinetics in the triple mutation of hDAT. As Y88 is spatially adjacent to R85 and K92 in helix TM1b, the double mutations on both Y88F and K92M are expected to induce more structural disturbance to the R85-D476 and K92-D313 salt brides, thus further decreasing Vmax of the transporter. Therefore, triple mutant Y88F/K92M/H547A has a lower Vmax compared to, single mutant K92M. Although including K92M mutation significantly decreases the Vmax for DA uptake in the triple mutant Y88F/K92M/H547A compared to Y88F/H547A, no significant change in the Bmax of [3H]WIN 35,428 binding is found in the double or triple mutants, suggesting that the multiple mutations may alter DAT re-uptake function rather than the substrate binding sites on DAT. This conclusion is also supported by the increased Km in Y88F/H547A and no change in Km in Y88F/K92M/H547A. However, single point mutations of Y88, K92, and H547 differentially affect the Vmax with no changes in the Km18. One possible explanation is that allosteric modulation of the dynamic DA transport process may be responsible for the enhanced Vmax and its associated DA uptake potency.According to our computationally modeled structures, Y548-Y470-Y551 is a stable motif (denoted as the YYY motif for convenience) essential for the conformational conversion of hDAT required for the DA transport process3H]DA to inhibit DA uptake relative to WT hDAT, which is similar to that in Y88F and H547A18. However, Y88F/H547A increased the DA uptake potency for cocaine and GBR12909, which is consistent with the single mutation of Y88F relative to WT hDAT17. Similar to Y88F17, Y88F/H547A displayed decreased potency for DA to inhibit [3H]WIN35,428 binding. However, the inhibition potency of [3H]WIN35,428 binding for cocaine was decreased in Y88F/H547A but increased in Y88F17. Y88F/H547A, similar to Y88F17, retained the [3H]WIN35,428 binding potency for GBR12909. GBR12909 is a selective inhibitor of DA uptake, binding to the piperazine acceptor site without influencing DA release42; while cocaine is a competitive inhibitor for DAT and prefers to an outward-facing transport state, leading to a decrease in DA transport44. According to our computational modeling prediction, both hDAT residues Y88 and H547 are the critical binding sites on DAT for Tat20. Therefore, our findings demonstrate that through allosteric modulation, mutation of Y88 and H547 alters the inhibition potency for DA uptake and DAT binding site for substrate and DAT inhibitors40.We further determined the effect of double mutant Y88F/H547A on the basal DA uptake via hDAT and binding potency for DA, GBR12909, and cocaine. Y88F/H547A had decreased the potency for WIN35,428 binding relative to WT hDAT. Second, Y88F/H547A augments basal DA efflux when compared to WT hDAT, which opposes our previous reports showing that neither Y88F nor H547A affected the basal efflux18. The elevated basal DA efflux in Y88F/H547A may be attributed to its elevated Vmax of DA uptake and DA accumulation. Taken together, Y88F/H547A, via an alteration of DAT conformational transitions, may influence the binding structure of hDAT-Tat complex, thereby disrupting Tat-induced inhibition of DA uptake. Studying the multiple mutation of DAT residues in Tat-DAT intermolecular interaction will greatly contribute to our ongoing proof of concept studies using novel allosteric modulators to establish their potential for therapeutic application in HAND.A major finding is that the double mutant Y88F/H547A completely attenuates the Tat-mediated inhibition of DAT reuptake function, which is consistent with our previous reports on Y88F- or H547A-induced attenuation of the Tat’s effect49. Our computational modeling has demonstrated that the dynamic DA uptake process includes outward-open, outward-occluded, and inward-open states21, which can be reversed by DA substrate and DAT mutation51. The current results indicate that the potency for AMPH inhibition of [3H]DA uptake was not altered in Y88F/H547A compared with WT hDAT, suggesting that the mutation of Tyr88 and His547 does not alter the interaction of AMPH with hDAT. However, AMPH-mediated DA efflux was reduced in H547A and Y88F/H547A, suggesting that mutations of Tyr88 and His547 residues alter the conformational equilibrium of the DAT transport transitions. Furthermore, AMPH decreases the Vmax of DA uptake in both WT hDAT and Y88F/H547A, which is consistent with the previous report51, however, the magnitude of the AMPH-mediated reduction of the Vmax in Y88F/H547A was greater than that in WT hDAT. Interestingly, the AMPH-mediated reduction of the Vmax was accompanied by increased Km values in WT hDAT but not Y88F/H547A. Given that DAT-mediated DA transport is accompanied by reuptake of two Na+ and Cl− ions, AMPH influx would transport Na+ through DAT and enhance its levels at the inner face of the transporter52. Therefore, AMPH decreases DA transport but increases Km values in WT hDAT, whereas altered transporter conformation by Y88F/H547A may attenuate the increased Km. Taken together, the results along with attenuated zinc-regulated [3H] WIN35,428 binding and increased basal DA efflux in Y88F/H547A further support that mutations of Tyr88 and His547 cause an alteration in conformational transitions, thereby disrupting the inhibitory effect of Tat on DAT reuptake function.AMPH, a substrate of DAT, competitively inhibits the reuptake of DA and elicits DAT-mediated DA efflux by reversal of the transporter18. An allosteric modulatory mechanism may contribute to Y88F/H547A-mediated attenuation of Tat-induced inhibition of DA transport. Understanding the multiple mutation effects of the identified DAT residues might provide a basis for a novel approach for developing compounds to attenuate Tat binding to DAT by an allosteric mechanism in HIV-1-infected individuals.In conclusion, our data further demonstrate DAT Tyr88 and His547 as important residues for DAT function and the Tat-DAT interaction. Double mutation of these residues preserves single mutant H547A-mediated enhancement of DA uptake and diminishes Tat-induced inhibition of DA transport53 and the constructed structure of hDAT-DA complex21. Protein docking and molecular dynamics simulations were employed to identify the conformation of hDAT-Tat complex. The energy-minimized complex structure used in this work were extracted from long-time equilibrated molecular dynamics simulation trajectories in our previous work35.We performed computational modeling and simulations to model the binding structure of hDAT with HIV-1 clade B type Tat based on the nuclear magnetic resonance (NMR) structures of Tat18. Single mutants, Y88F, K92M, and H547A, are expected to abolish hydrogen bonds between hDAT and Tat. Double (H547A/Y88F) and triple (H547A/Y88F/K92M) mutations were generated based on the wild type hDAT (WT hDAT) sequence  by site-directed mutagenesis. We then used the synthetic cDNA encoding hDAT subcloned into pcDNA3.1 + as a template to generate mutants using QuikChange™ site-directed mutagenesis Kit . We performed the DAT sequence for confirming the sequence of the mutant construct at University of South Carolina EnGenCore facility. Plasmids DNA were propagated and purified using a plasmid isolation kit .We selected the mutations of Y88, H547, Y88/H547 and Y88/K92/H547 based on predictions of computational modeling and simulations Fig.  and our 2 incubator. Once cells were growing to 100% confluence on plates, cells were seeded at a density of 1 × 105 cells/cm2 in 24-well plates and transfected with WT hDAT or mutants by Lipofectamine 2000 (Life Technologies). Twenty-four hours after transfection, intact cells or cell suspensions were used for experiments.In the current study, we used rat pheochromocytoma cells , Manassas, VA), which were maintained in Dulbecco’s modified eagle medium  supplemented with 15% horse serum, 2.5% bovine calf serum, 2 mM glutamine and antibiotics, 100 U/mL penicillin and 100 µg/mL streptomycin. Cells were cultured at 37 °C in a 5% COmax) or Michaelis-Menten constant (Km) of [3H]DA uptake in intact PC12 cells transfected with WT hDAT or mutants as reported previously17. Twenty-four hours after transfection, cells in 24-well plates were washed twice with Krebs-Ringer-HEPES (KRH) buffer  and incubated with nomifensine  as nonspecific uptake for 10 min at room temperature. A fixed concentration of [3H]DA  mixed with one of six concentrations of unlabeled DA  were applied to cells at room temperature for additional 8 min. The specific DAT-mediated DA uptake was calculated by subtracting non-specific uptake from total uptake (in the absence of nomifensine). The reaction was terminated by removal of solution from wells and quickly washed three times with ice cold KRH buffer. Cells were then lysed in 500 μL of 1% SDS for an hour and radioactivity was measured using a liquid scintillation counter .In order to determine whether DAT mutants alter DAT function, we evaluated the maximal velocity  as the previous reports30. Intact PC12 cells transfected with WT or Y88F/H547A were preincubated with or without PMA for 30 min at 37 °C followed by additional 8 min incubation with six concentrations of mixed [3H]DA as described above. To determine the effect of amphetamine (AMPH) on Y88F/H547A-mediated transporter conformational transitions, Vmax or Km of [3H]DA uptake in WT and Y88F/H547A was determined in the presence or absence of 1 µM AMPH  as reported previously51.To confirm whether Y88F/H547A-induced increase in V2+-regulation of DA transport, we performed the competitive inhibition of DA uptake in intact PC12 cells transfected with WT or mutant, which were preincubated with a series of final concentrations of DA (1 nM-1 mM), GBR12909 (1 nM-10 µM), cocaine (1 nM-1 mM), AMPH (1 nM-1 mM) or ZnCl2  at room temperature for 10 min followed by additional 8 min incubation with a fixed concentration of [3H]DA .To determine whether these mutations alter the affinity of DAT substrate or inhibitors as well as Zn3H]DA uptake in PC12 cells transfected with WT or mutant in the presence or absence of Tat protein. In brief, cells were dissociated with trypsin/EDTA  and resuspended in culture medium and incubated at room temperature for 10 min. The dissociated cells were collected by centrifugation at 400 × g for 5 min at 4 °C and washed once with phosphate-buffered saline followed by additional 5 min centrifugation . Finally, the resulted cell pellets were resuspended in KRH assay buffer. The cell suspensions from WT hDAT or Y88F/H547A mutant were then preincubated with or without recombinant Tat1–86  at room temperature for 20 min followed by additional 8 min incubation with a mixed [3H]DA uptake . Non-specific [3H]DA uptake was determined in the presence of 10 µM nomifensine. The update reaction was terminated by immediate filtration through Whatman GF/B glass filters (presoaked with 1 mM pyrocatechol for 3 h) followed by three washes with 3 mL of ice-cold KRH buffer containing pyrocatechol using a Brandel cell harvester . Radioactivity was determined as described above.In order to determine whether double mutation of Y88 and H547 disrupts Tat and DAT interaction, we performed the specific [max or Kd) of [3H]WIN 35,428 binding in intact PC12 cells transfected with WT hDAT or mutant. Cells were washed with sucrose-phosphate buffer twice  and then incubated with one of the six concentrations of [3H]WIN 35,428  in a final volume of 500 μL on ice for 2 h. Nonspecific binding at each concentration of [3H]WIN 35,428 was determined by adding in the presence of 30 µM cocaine  and subtracted from total binding to obtain the specific binding. For competitive inhibition experiments, assays were performed in a final volume of 250 μL. Intact PC12 cells transfected with WT or mutated hDATs were incubated in buffer containing 25 μL of [3H]WIN 35,428  and one of ten concentrations of DA (1 nM- 100 μM), cocaine (1 nM- 100 μM), GBR12909 (0.01 nM- 1 μM) or ZnCl2  on ice for 2 h. Assays were terminated by removing reaction reagents in each well followed by two washes with ice-cold assay buffer. Cells were lysed with 1% SDS for an hour and subjected to the radioactivity measurement as described above.Binding assays were used to determine the kinetic parameters  were lysed rapidly in 1% SDS after preloading with [3H]DA. To collect factional efflux samples, buffer (500 μL) was added into a separate set of cell wells and transferred to scintillation vials after 1 min as an initial fractional efflux, and another 500 μl buffer was added to the same wells and collected after 10 min as second fractional efflux. Additional fractional efflux at 20, 30, 40, 50 min, respectively, was repeated under the same procedure. After last fractional efflux, cells were lysed and counted as total amount of [3H]DA remaining in the cells from each well.Basal DA efflux was performed at room temperature as described previously51. PC12 cells transfected with WT hDAT and its mutants growing in 24-well plates were preloaded with [3H]DA  for 20 min at room temperature. Subsequently, cells in all plate wells were washed with KRH for three times, cells were then incubated with KRH buffer containing AMPH (5–250 µM) or vehicle  for 15 min at room temperature. After incubation, the buffer solution incubated with AMPH or vehicle was collected as AMPH-stimulated efflux or baseline efflux. Cells were lysed in 1% SDS and subject to count for obtaining the initial amount of DA  in the cells.To further determine DAT mutation-mediated transporter confirmation transitions, basal efflux and AMPH-stimulated DA efflux in WT hDAT and its mutants were measured as reported previouslyn as the number of independent experiments for each experiment group. Pharmacological parameter values  and IC50 from [3H]DA uptake and [3H]WIN 35,428 binding were calculated by nonlinear regression analysis using a one-site model with variable slope. To determine the significant difference in the pharmacological parameters between unpaired sample groups, such as WT hDAT and its mutants, unpaired Student’s t test was used for the statistical comparisons. Significant differences between sample groups were analyzed with separate ANOVAs followed by post-hoc, Bonferroni multiple comparison test as indicated in the results Section of each experiment. All statistical analyses were performed using IBM SPSS Statistics version 25, and differences were considered significant at p < 0.05.In the current study, all data are presented as mean ± SEM with


Prevailing agro-ecological conditions and intermingling of human and animals in intensive farms in urban and peri-urban areas in Africa favour cross species transmission of pathogens at the human-animal interface. However, molecular epidemiology studies of zoonotic swine influenza viruses in this region are limited. In this study, isolates of pandemic influenza virus (H1N1pdm09) obtained from pigs in Nigeria were fully sequenced. BLAST of swine influenza virus genes from Nigeria was carried out in GenBank and gene alignment was done using MEGA version 7. Maximum likelihood method (PhyML program) was used to determine gene evolutionary relationships with other viruses and phylogenetic trees were constructed to infer genomic clusters and relationship. Swine influenza viruses isolated and sequenced in this study were monophyletic and 99% congenetic with human isolates from Nigeria, Cameroon, Ghana and USA suggesting reverse zoonotic transmission from humans to pigs in intensive husbandry. A Q240R and S31N substitution among others were detected in the haemagglutinin and matrix genes, respectively, indicating potentials for mutations during interspecies co-mingling and transmission. The A/H1N1pdm09 viruses circulating in pigs that are also exposed to avian influenza in the same epidemiological zones could engender emergence of novel viruses with zoonotic or pandemic potential requiring enhanced surveillance and monitoring. Pigs are mixing vessels of influenza viruses and known reservoirs of virus genes capable of causing emergence of novel influenza virus strains with potential adverse economic and public health consequences . AlthougThe pandemic H1N1pdm09 has been shown to be a product of gene reassortment of avian, swine and human influenza viruses . The ecoInfluenza virus surveillance at the human-animal interface was carried out between July 2010 and June 2012 in a multi-complex commercial piggery operation located in a peri-urban zone in Lagos, southwestern Nigeria. Samples were collected from 227 pigs showing upper respiratory signs compatible with influenza-like infection and were screened for the presence matrix gene by real-time RT-PCR . The stuRT-PCR positive samples were also subtyped as 2009 pandemic A/H1N1 with subtype-specific primers and probes. Each gene segments were further amplified using several PCRs targeting overlapping fragments with cycling conditions set at 95°C for 2 min, 95°C for 45 s, 46°C for 1 min, 72°C for 4 min, 72°C for 10 min and 4°C  for 40 cycles . . Cycle condition for sequence reaction was set at: 96°C for 10 s, 50°C for 5 s and 60°C for 4 min for 25 cycles. This was followed by gel filtration clean up of sequenced products using Column AutoSeq G-50 before analysis in 16 capillary Sequence Analyser . Subsequently, sequence assembly and editing were performed using Seqscape version 2.5 . Gene sequences were thereafter compared with corresponding genes of other influenza virus strains obtained from GenBank and Global Initiative on Sharing All Influenza Data (GISAID). These include human influenza sequences from Nigeria, Ghana and Cameroon, viz.: A/Nigeria/4280/2011, A/Ghana/601/2011 and A/Cameroon/EID/07/11/1870/2011.To infer the evolutionary relationship for each gene segment, we employed maximum likelihood (ML) methods available through the PhyML program, incorporating a GTR model of nucleotide substitution with gamma-distributed rate variation among sites and a heuristic SPR branch-swapping search Parameter values for the GTR substitution matrix, base composition, gamma distribution of the rate variation among sites (with four rate categories) and proportion of invariant sites (I) were estimated directly from the data using MEGA 7. A bootstrap resampling process  using the neighbor-joining (NJ) method and incorporating the ML substitution model defined above, was employed to assess the robustness of individual nodes of the phylogeny . Nucleotwww.ncbi.nlm.nih.gov). The pandemic Swine influenza virus that were isolated from the piggery complex in Nigeria and used in the phylogenetic study has earlier been reported . The GenTo gain more information about the phylogenetic relatedness of the sequenced viruses in this study, sequences from GISAID platform were retrieved and compared. The HA gene of A/swine/Nigeria/12VIR4047/2011 and the other Nigeria swine isolates are closely related to A/Nigeria/4280/2011, A/Ghana/601/2011 and A/Cameroon/EID/07/11/1870/2011 deposited in GISAID, all of which are human isolates of influenza H1N1pdm09 virus clustering together in the phylogenetic tree . HoweverSequence analysis of the NA gene relative to A/California/07/2009 also revealed amino acid substitutions at positions V106I, N248D, Y351F, S439G. S439G substitution in the amino acid is also unique to A/Swine/Nigeria/VIR44-09/2009. All other reference viruses has S in this position. The PB2 protein of Nigerian isolate of H1N1pdm09 had D527G amino acid substitution, for PB1 G replaced K in position 736, for NS gene amino acid substitutions between A/Swine/Nigeria and prototype A/California include G28V, W103C, Q109K, I111M, I123V, W187E, G189D, V234M, S246N. The PA gene also had four substitutions presented in https://platform.gisaid.org/epi3/frontend#312740). That sample was traced to Kano in Northern Nigeria, a major commercial center with direct human and material traffic with Lagos in the southern part of the country where this study was carried out. Similar cases and detection of H1N1pdm09 in human were also reported in Nigeria about the same period (2009 to 2011) suggesting prior circulation of the virus in humans before probable transmission of the virus to pigs [The full genome of three H1N1pdm09 isolates from pigs in Nigeria that were analysed in this study were most similar to influenza H1N1pdm09 sequences from human hosts as obtained in GenBank BLAST. This is also supported phylogenetically with the close similarities of A/swine/Nigeria/12VIR4047/2011(H1N1) with A/Nigeria/4280/2011(H1N1), A/Ghana/601/2011(H1N1) and A/Cameroon/EID/07/11/1870/2011(H1N1) which are viral sequences from human. Ghana and Cameroon are neigbouring West African countries with social and economic linkages as well as human/material traffic with Nigeria, hence the potential for transboundary transmission of infectious pathogens. Interestingly, earlier detection of H1N1pdm09 influenza virus in pig was reported in Cameroon [et al., 2000). Most H1N1pdm09 influenza virus circulating globally including those detected in human host in Nigeria have Q in position 240 (Q226 -H3 numbering) [The observed transmission and circulation of influenza H1N1pdm09 from humans to pigs resulted in the emergence of a virus with few amino acid substitutions in the animal host. These substitutions include the 240 position (H1 numbering) of H1N1pdm09 HA which is occupied by either amino acid glutamine (Q) in 95% of cases including in H1, H2 and H3 or arginine (R) in only about 5% of cases (Matrosovich mbering) . This ismbering)  where huThough the observation of host-induced selection of variants was reported for Q226R or D225G of earlier human H1N1pdm09 viruses, this study observed Q240R substitution. Previous studies found four important positions  to be involved in the specific binding capacity of HA to the host cell receptor . Thus thThis first and till date only report of complete genome sequencing of H1N1pdm09 virus in Nigeria represent the first full data in the GenBank from Africa and provides baseline genomic data on swine influenza virus circulating in pigs in Nigeria and probably the rest of West African agro-ecological zone as later reported in Kenya, Togo, Uganda and further detection in Nigeria and Ghana –19. ThisGiven the genetic characteristics of influenza viruses, the continued circulation of H1N1pdm09 viruses in pigs in Nigeria and other parts of West Africa provide opportunities for antigenic drift and shift and co-circulating with human, avian or swine influenza viruses. This increases the risk of emergence of influenza virus variants with enhanced intra and interspecies transmission and pathogenicity in a region recognized as a zoonotic hotspot. This thus warrants continued ecological and molecular surveillance.


Microbacterium sp. QH-2 was isolated in this study. Scanning electron microscopy (SEM) images showed protrusions on the bacterial surface of strain QH-2 after an 18 h incubation in media under 10 mM Cr(VI) treatment. Results of the experiments on the capacity of reducing Cr(VI) indicated that strain QH-2 could reduce 100% Cr(VI) less than 48-96 h. When media with 4 mM Cr(VI) were incubated, the fastest reduction rate of strain QH-2 could come up to 2.17 mg/L Cr(VI) h−1. Furthermore, strain QH-2 could reduce Cr(VI) over the pH between 7 and 10. The optimum pH to reduce Cr(VI) by strain QH-2 was 9. Strain QH-2 also exhibited a relatively high tolerance even to 20 mM Cr(VI). These results declared that strain QH-2 had the potential to detoxify Cr(VI) in the Cr(VI)-contaminated soil or effluent.The biotransformation of hexavalent chromium [Cr(VI)] via Cr(VI)-reducing microorganisms is considered an ecofriendly approach to detoxify Cr(VI). A new Cr(VI)-reducing bacterium named In recent years, because of its use in pigments, ceramics, leather tanning, metal corrosion inhibition, and refractory materials, environmental pollution caused by chromium (Cr) in soil and sediments, etc. is widespread , 13. AccExcept for Cr(VI), trivalent chromium [Cr(III)] is also a major present form in the environment. Cr(III) has a low solubility. In addition, it can act as a nutrient for organisms and microorganisms , 18. By 0) [At present, the chemical reduction is the widely used method to reduce the toxicity of Cr(VI). Common chemical reductants include ferrous sulfate  and elem0) . Additio0) , biochar0) , and mod0)  can also0) . Therefo Cellulosimicrobium sp. [ Stenotrophomonas maltophilia [ Alishewanella sp. WH16-1 [ Pisolithus sp1 [ Penicillium oxalicum SL2 [The biotransformation of Cr(VI) is considered to be capable of being performed by Cr(VI)-reducing bacteria and fungi, which can be isolated under aerobic and anaerobic conditions. Various Cr(VI)-reducing bacteria such asbium sp. , Stenotrtophilia , and Ali. WH16-1  have beethus sp1  and Peniicum SL2  can reduicum SL2 .In present study, we aim to (i) isolate a new Cr(VI)-reducing bacteria from Cr(VI)-contaminated alkali soil under aerobic conditions, (ii) study the morphological changes of the bacterial strain under Cr(VI) and the phylogenetic tree analysis, and (iii) evaluate both the capacity to reduce and resist Cr(VI) by the bacterial strain.2CrO4 under aerobic conditions at 30°C for 1 week. During the incubation period, sterile water was infused to keep the soil hydrated. After the incubation, 10 g of soil was added to 100 mL of 0.85% NaCl solution then shaken at 30°C for 2 h. After the shaking process, the soil extract was taken. The soil extract was serially diluted, then spread on Luria-Bertani (LB) plates with K2CrO4 [Soil samples were collected from the alkali soil contaminated with Cr(VI) because of the stacking of Cr slag in Qinghai Province, China. The pH of the soil (pH 9.85) was measured by using a soil to water ratio of 1:2.5. Total Cr in soil was 2267.1 mg/kg. To enrich Cr(VI)-reducing bacterial strains, 100 g of soil was incubated with 5 mM Kh K2CrO4 . Then, LDNA was extracted via the FastDNA Spin Kit based on the description then dissolved in Tris-EDTA buffer. Then the mixture was stored at -20°C before PCR amplification. The 16S rRNA genes were amplified with 27F and 1492R . ConditiThe bacterial strain was inoculated in the fresh LB media with 10 mM Cr(VI) for the study of the influence of Cr(VI) to the cell morphology via SEM. The media were incubated for 18 h . The bacterial cells were centrifuged . After the centrifugation, cells were taken then washed with phosphate buffer saline (PBS) for three times (wash every fifteen minutes). After the washing process, cells were fixed with 2.5% glutaraldehyde then stored at 4°C. Before the SEM, the glutaraldehyde should be removed from the cells. Hence, the cells were washed with ultrapure water for three times. Then, the bacterial cells were dehydrated with 50%, 70%, 85%, 95%, and 100% (V/V) ethanol and dried. Finally, the cell samples were coated with gold and the morphology of the cell samples was examined with SEM .600) via the spectrophotometer . The culture supernatant was taken and then passed through a 0.22 mm filter for detecting the amount of Cr(VI) remaining in media via the 1,5-diphenylcarbazide (DPC) method at 540 nm . In brief, sulfuric acid (H2SO4) and phosphoric acid (H3PO4) were diluted with ultrapure water , respectively. Then 0.2 g of DPC was dissolved in 50 mL acetone and diluted with ultrapure water to 100 mL. This solution was used as the chromogenic reagent. The 0.2829 of K2Cr2O7 (dried at 110°C for 2 h) was dissolved with ultrapure water in a 1000 mL volumetric flask which was used as the Cr (VI) stock solution. The Cr (VI) standard solution was by appropriate dilution of the Cr (VI) stock solution mixed with 0.5 mL dilute H2SO4, 0.5 mL dilute H3PO4, and 2 mL chromogenic reagent. The culture supernatant of sample was centrifuged then mixed with the same solutions above in a 50 mL colorimetric tube. The mixture was measured at OD540 and the ultrapure water was used as a reference. Remaining Cr(VI) was considered to reflect the capacity for Cr(VI) reduction and calculated as 0 is the Cr(VI) in controls.In order to study the Cr(VI)-reducing capacity of the bacterial strains, 10 mL of logarithmic phase bacterial cultures grown in different initial concentrations of Cr(VI) (1 mM - 4 mM) was injected into the fresh LB media . Fresh LB media supplemented with the same initial Cr(VI) concentrations, but not inoculated with the bacterial cultures, served as controls. Culture media were incubated . Samples of culture media were aseptically withdrawn at different time point then centrifuged  to detect the optical density at 600 nm  to detect the remaining Cr(VI).Batch tests were performed to discuss the influence of different pH to reduce Cr(VI). Fresh LB media were adjusted to targeted pH  using HCl or NaOH. Then, the media were inoculated with logarithmic phase bacterial cultures and supplemented with 5 mM Cr(VI). Culture media were kept in a rotatory shaker . Noninoculated controls adjusted to pH and supplemented with Cr(VI) were incubated in the same conditions. Samples of culture media were collected at different intervals to detect the OD600 was monitored at different intervals. Inoculated media without Cr(VI) served as controls.For the evaluation of resistance of bacterial strains to Cr(VI), logarithmic phase bacterial cultures and different concentrations of Cr(VI)  were added to the fresh LB media (pH 9.0). Culture media were kept in a rotatory shaker with the same conditions as described above. The OD Microbacterium species in the BLAST programme, and some reported strains that can reduce Cr(VI) were selected to establish the phylogenetic tree via MEGA 4.0 software.The nucleotide sequence of strain QH-2 was submitted to GenBank (MH319855.1). Then the nucleotide sequences of strain QH-2, someExperiments of this study had three replicates in order to calculate the standard deviations. Microbacterium genus. The neighbour-joining phylogenetic tree of strain QH-2 is depicted in  Microbacterium sp. strain CHQ-1 (KX618653.1) and Microbacterium sp. Z5 (HM171926.1), etc. Consequently, strain QH-2 could be characterized and named as Microbacterium sp. QH-2 (MH319855.1).A number of bacterial strains were isolated through aerobic cultivation from several single colonies in this study, and the strain QH-2 which had a strong capacity for reduction Cr(VI) was selected. Based on BLAST analysis, strain QH-2 belonged to theThe SEM images of strain QH-2 from the media supplemented with 10 mM Cr(VI) and without Cr(VI) are exhibited . Strain −1 under 4 mM Cr(VI) in media.Selected pH of 9.0 used in this study was the optimum pH to grow and chosen according to the growth curves performed at different pH (Supplementary The influences of pH (6 to 10) on the reducing Cr(VI) via strain QH-2 are showed in As shown in Microbacterium sp. QH-2 belongs to a new Cr(VI)-reducing bacterium that was isolated from the alkali soil contaminated with high concentration of Cr(VI). The soil is used for stacking Cr slag all the year round. This soil environment also determines that strain QH-2 has high resistance to Cr(VI). When strain QH-2 was incubated in the media supplemented with 10 mM Cr(VI) (M Cr(VI) , the pro Microbacterium have been reported to have the capacity to reduce Cr(VI). For instance, Microbacterium MP30 could reduce 0.1 mM Cr(VI) less than 30 h with a reduction rate of 0.17 mg/L Cr(VI) h−1 [ Microbacterium Cr-K29 and Microbacterium Cr-K20 had reduction rates of 2.17 and 0.52 mg/L Cr(VI) h−1, respectively [ Microbacterium culture (X7) could reduce 100 mg/L Cr(VI) when media were under the incubation after 75 h (1.33 mg/L Cr(VI) h−1) [ Microbacterium species.Members of the genus(VI) h−1 . When 50ectively . MicrobaVI) h−1) . Accordi Pseudochrobactrum asaccharolyticum (KC618329) [ Bacillus sp. MDS05 (EU236673) [ Microbacterium sp. (JN674183) [ Bacillus amyloliquefaciens (JQ429762). Das et al. [ Bacillus amyloliquefaciens (JQ429762) could reduce only 56.5% of nearly 4 mM Cr(VI) after 144 h. By contrast, strain QH-2 could almost completely reduce 4 mM Cr(VI) after 96 h (Compared with the reduction rates of some species of Cr(VI)-reducing bacterial strains such asC618329) , BacilluN674183)   enhanced with the increase in the pH from 6 to 9 [The pH value is an important factor to have influence on reducing Cr(VI). When the media were incubated after 108 h, 5 mM Cr(VI) was almost 100% reduced in the media at the initial pH of 8 and 9, while residual Cr(VI) was still existing at the initial pH of 7 and 10 . Sayel em 6 to 9 . These d Leucobacter sp. CRB1 under pH 9 [ Bacillus amyloliquefaciens showed the best effect of reducing Cr(VI) at pH 7 [ Bacillus sp. was 8. In addition, Enterococcus gallinarum had the most obvious Cr(VI) reduction effect at pH 10 [ Bacillus sp. strain KSUCr5 [In this study, the optimum pH to reduce Cr(VI) for strain QH-2 is 9. Similarly, all the Cr(VI) was reduced by at pH 7 . Cheng a at pH 7  found that pH 10 . Furthern KSUCr5 . The eff6+-induced oxidative stress by activating reactive oxygen species scavenging enzymes and DNA repair, etc. [Strain QH-2 also exhibited a relatively high tolerance even to 20 mM Cr(VI) . The resir, etc. , 45. How Microbacterium sp. (JN674183) was isolated from the medium supplemented with 19.2 mM Cr(VI). This meant that Microbacterium sp. (JN674183) could resist 19.2 mM Cr(VI). When compared with other strains, strain QH-2 exhibited a higher tolerance to Cr(VI), even at 20 mM. Although Bacillus sp. MDS05 (EU236673) could resist 48 mM Cr(VI) [ Bacillus sp. MDS05 (EU236673) was lower than that of strain QH-2. These results indicate that strain QH-2 could exist in environments polluted with high concentrations of Cr(VI). Meanwhile, strain QH-2 has potential for the bioremediation of Cr(VI) in soil or effluent.As shown in M Cr(VI) , its cap Microbacterium sp. QH-2 has a fast reduction rate and relatively high tolerance to Cr(VI). These results mean that Microbacterium sp. QH-2 might have enough potential to detoxify Cr(VI), especially in Cr(VI)-contaminated alkali soil. Further studies will investigate the biological reduction mechanism and the resistance mechanism to Cr(VI) by Microbacterium sp. QH-2 and study the detoxification effects of Microbacterium sp. QH-2 in Cr(VI)-contaminated soil or effluent.The bioremediation via Cr(VI)-reducing bacteria is a promising approach. The findings presented here indicate that


Western blotting analysis showed that each epitope interacted with the positive serum of sheep that had been naturally infected with CCHFV. Amino acid sequence alignment between each epitope and their homologous proteins showed that they were almost 100% conserved among 12 CCHFV sequences from different lineages, except for epitopes E1a, E1b and E2. Three-dimensional structural modeling analysis showed that all identified epitopes were located on the surface of the NP “head” domain. This study identified fine epitopes on the N- and C- terminals of NP, which will increase the understanding of the structure and function of NP, and it could lay the foundation for the design and development of a CCHFV multi-epitope peptide vaccine and detection antigen.Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne pathogen that causes severe disease in humans. CCHFV is widely distributed in more than 30 countries and distinct regions, which means that it poses a serious threat to human health. The nucleocapsid protein (NP) encoded by the CCHFV S gene is the primary detectable antigen in infected cells, which makes it an important viral antigen and a clinical diagnostic target. In this study, the modified biosynthetic peptide (BSP) method was used to identify the fine epitopes on the N- and C- terminals of NP from the CCHFV YL04057 strain using rabbit antiserum against CCHFV-NP. Nine epitopes were identified: E1a ( Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease that was first discovered in the West Crimean Peninsula in 1944. It has been reported in more than 30 countries and distinct regions in Central and Southern Africa, Southeast Asia, Southeast Europe and the Middle East, with case fatality rates of up to 30–50% . CCHF isOrthonairovirus in the family of Nairoviridae  of the NP “stem” domain (NP2) were identified using rabbit serum against recombinant (r)-NP combined with the biosynthetic peptide (BSP) method . HoweverThe study was approved by the Committee on the Ethics of Animal Experiments of Xinjiang Key Laboratory of Biological Resources and Genetic Engineering (BRGE-AE001), Xinjiang University. The animal serum samples were collected using random sampling and this process did not involving killing the animals.NP1 (aa 1–200), pGEX-KG-NP2 (aa 170–305) and pET-32a-NP3 (aa 286–482) were previously constructed and stored by this research group [Escherichia coli BL21 (DE3) competent cells were used to express 16/8mer peptides fused with a truncated GST188 protein  [pET-32a-ch group . The proch group . The serch group . In our ch group . Serum s of GST) . Goat anBamH I and Sal I , E. coli strain BL21 (DE3) competent cells , QIAquick Gel Extraction Kit , unstained or prestained molecular weight markers , 0.2 μm nitrocellulose membrane , and enhanced chemiluminescence (ECL) plus western blotting detection kit  were obtained. Other general chemicals were obtained from Shanghai Sangon Co., Ltd (China).DNA ligase and restriction enzymes To map epitopes on the NP1 and NP3 segments, we used the feasible strategy shown in BamH I and TAA-Sal I sites at the 5`- and 3`- ends were synthesized by SBS Genetech Co., Ltd . Each r-plasmid expressing a 16/8mer peptide was constructed according to the GST188-BSP method [BamH I and Sal I; iii) transforming E. coli BL21 (DE3) competent cells with the ligation mixture; iv) screening of the r-clones by carrying out sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using total proteins from each induced clone and observing whether there is a specific 16/8mer peptide on the gel; and v) sequencing of inserted DNA fragment encoding each 16/8mer peptide for each determined r-clone to ensure that all synthesized DNA sequences are accurate.All plus and minus strands of DNA fragments that encoded target 16/8mer peptides and had cohesive end nucleotides of P method , in whicE. coli BL21 (DE3) cells, which was fused with the GST188 protein [600) of 0.5–0.7, and then grown at 42°C for 4 h to induce the expression of the target short peptide. All collected cell pellets containing the expressed 16/8mer peptide fusion proteins were stored at -20°C.Each above-determined r-clone was used to express a 16/8mer peptide in The cell pellets obtained from 2 mL culture of expressed 16/8mer peptide were boiled at 95°C in 200 μL of 1×SDS-PAGE loading buffer for 10 min, and the proteins were resolved by 15% SDS-PAGE under reducing conditions. Two gels were obtained for each16/8mer. One gel was stained with Coomassie brilliant blue G-250 for analyzing the bands corresponding to the target 16/8mer peptide. The other gel was used for western blotting by electrotransferring the proteins onto a 0.2 μm nitrocellulose (NC) membrane . RegardiTo assess the conservation of each identified epitope among CCHFV homologous proteins, 12 NP aa sequences from different countries and genetic lineages were obtained from GenBank based on the phylogenetic tree of CCHFV strains . As far In a previous study, the truncated NP1 and NP3 segments of CCHFV-NP were expressed using the prokaryotic expression vector pET-32a, and each predicted protein was fused with Trix tag, S tag and His tag (with a size about 18 kDa) . HoweverE. coli [To determine the existence of epitopes on the NP1 and NP3 segments, both NP1 and NP3 were truncated into 24 16mer BSPs . The 48 overlapping 16mer BSP sequences and the corresponding sites on NP are shown in E. coli , and theE. coli  and 184GGDENP189 (E1b), based on the shared residues (352PLKWGKK358 (E2) in P33, 363FADDS367 (E3) in P34, 399NPDDAA404 (E4) in P38 and P39, 447DIVASEHL454 (E5a) and 452EHLLHQSL459 (E5b) in P45, 464SPFQNAY470 (E6) in P47, and 475NATSANII482 (E7) in P48. Thus, two specific epitopes were identified on the NP1 segment (NP1-200) and seven specific epitopes were identified on the NP3 segment (NP286-482). None of the 8mer peptides reacted with the CCHFV-negative rabbit sera (data not shown). The sensitivity of the antigen-antibody reaction was determined using the same quantity of peptides for the western blotting detection, and the data were quantitatively analyzed using Image-J software (http://rsb.info.nih.gov/ij/). The relative grayscale results showed that 8mer peptides P57, P58, P87 and P106 had a significantly (P<0.05) higher sensitivity for the antigen-antibody reaction  skimmed milk powder, incubated sequentially with anti-CCHFV sheep serum (1:100 dilution) and mouse anti-goat IgG (1:5000 dilution), and then visualized by ECL. All the selected 8mers were recognized by the serum of sheep with a confirmed history of CCHFV infection as the primary antibody , while n™ software [178NLILNRGG185), E2 (352PLKWGKK358), E3 (363FADDS367), E4 (399NPDDAA404), E5a (447DIVASEHL454), E5b (452EHLLHQSL459), E6 (464SPFQNAY470) and E7 (475NATSANII482) are the eight epitopes that are located on the "head" surface of the NP protein (184GGDENP189) is not labeled in PyMOLsoftware  was used protein . This is protein . As the 1-200 and NP286-482 from the Chinese CCHFV strain YL04057 (ACM78470.1) and 11 other strains from different countries and lineages (178NLILNRGG185) was conserved in all CCHFV strains except the South African strain assigned to lineage Africa 3 (AAZ38665.1), in which L179 was substituted with Q179. The E1b motif (184GGDENP189) was conserved in all CCHFV strains except the Chinese strain assigned to lineage Asia 2 (ADD64468.1), in which N188 was substituted with S188. To further determine whether the epitope peptides with residue differences in 1E1a, NQILNRGG; v2E1a, NLFLNRGG; v3E1a, NLILNRGS; v4E1b, GSDENP; v5E1b, GGDESP; and v6E2, PLKWGKK) and explored their antigenic properties. Specifically, single aa substitutions were made within E1a , E1b (G185S and N188S) and E2 (L353M). The results show that the two BSPs with one variable residue v1E1a (L179Q) and v5E1b (N188S) strongly reacted with rabbit pAbs against CCHFV-NP, but the others had no reactivity . Although these studies confirm that the central region of CCHFV-NP is highly antigenic, the N- (NP1-200) and C-terminals (NP286-482) of CCHFV-NP also have antigenicity , E1b (184GGDENP189), E2 (352PLKWGKK358), E3 (363FADDS367), E4 (399NPDDAA404), E5a (447DIVASEHL454), E5b (452EHLLHQSL459), E6 (464SPFQNAY470) and E7 (475NATSANII482).In this paper, to carry out a comprehensive linear epitope scan of CCHFV-NP, the modified epitopes of N-terminal NP1 and C-terminal NP3 segments were identified using a modified BSP method. First, we successfully synthesized 48 overlapping 16mer peptides and 58 overlapping 8mer peptides, and we identified nine minimal BCE linear motifs by western blotting with pAbs. The epitopes comprised E1a (182-195 reacted with 13 of 16 human serum samples and had high antigenicity. Our study identified the epitope E1b (184GGDENP189), which is consistent with results from Burt et al [The sequences of 12 strains of CCHFV from different countries and lineages were analyzed. The results showed that six epitopes, E3, E4, E5a, E5b, E6 and E7, were 100% conserved among the 12 CCHFV strains, while the epitopes of E1a, E1b and E2 showed homologies of 93.75%, 96.67% and 97.14%, respectively. Burt and colleagues  predictert et al .™ software. The results showed that only E1b (184GGDENP189) is located on unordered loop with no electron density, according to the 3D structural simulation analysis. The other eight motifs were exposed on the surface of the 3D structure, in the helix-angle-helix region Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 FigTo determine the sensitivity of the antigen-antibody reaction involving the 8mer peptides, quantitative analyses were performed using the same quantity of peptides for detection. The relative grayscale level of each 8mer peptide compared to the positive control, CP , was analyzed according to the results in (TIF)Click here for additional data file.S2 Fig179Q, I180F and G185S in E1a, G185S and N188S in E1b, and L353M in E2 were mutated to analyze the antigenicity. CK, negative control (GST188 protein expressed by pXXGST-3).According to the results of homologous analysis in (TIF)Click here for additional data file.S3 Fig183S), E1b (N188S) and E2 (L353M). GenBank code ACM78470.1 represents the CCHFV YL04057 strain.As far as we know, 11 complete nucleocapsid protein (NP) sequences of CCHFV strains isolated in China have been registered in the GenBank database. The 11 Chinese strains’ sequences corresponding to aa residues 1 to 200 of NP  and 286 to 482 of NP  were retrieved from GenBank for sequence alignment using the ClustalW program. The 11 strains showed good conservation at the E2, E3, F4, E5a, E5b, E6 and E7 sites. There was only differences in E1a (R(TIF)Click here for additional data file.