Agglutinins

The 327 Leptospira spp. used in this study are listed in Table S1. Genomic DNA was extracted as described previously [20] (link), with the exception of isolates from the National Center for Emerging and Zoonotic Infectious Diseases, Centers for Diseases Control and Prevention, USA, which were extracted with the addition of dimethyl sulfoxide. For isolates for which the species was unknown, identification was undertaken by amplification and sequencing of the near full-length 16S rRNA gene (rrs), as described previously [20] (link). Sequence data for rrs has been deposited in GenBank (see Table S2 for accession numbers). The serovars of isolates from recent human leptospirosis cases in Thailand, Laos and Sri Lanka were determined by the WHO/FAO/OIE Collaborating Center for Reference and Research on Leptospirosis, Queensland Health Forensic and Scientific Services, Brisbane, Australia using the cross-agglutinin absorption test (CAAT). The serovars of contemporary isolates from China were determined using CAAT at the China Medical Culture Collection of National Institutes for Food and Drug Control, China.
Ethical approvals to obtain contemporary clinical isolates of Leptospira spp. are as follows. Isolates from patients in northeast Thailand between October 2000 and December 2006 were obtained during a prospective study of acute febrile illness [20] (link), which was approved by the Ethical Committee of the Ministry of Public Health, Royal Government of Thailand. Isolates from patients in Laos between August 2006 and October 2009 were obtained during a prospective fever study approved by the Oxford Tropical Research Ethics Committee (OXTREC) and the National Ethics Committee for Health Research (NECHR), Ministry of Health, Government of Lao PDR. Isolates obtained from patients in Sri Lanka between June 2006 and May 2007 were obtained during a prospective fever study approved by the Research Ethics Committee of the Liverpool School of Tropical Medicine, UK and the Ethical Review Committee of the Faculty of Medicine of the University of Kelaniya, Sri Lanka. Written informed consent was obtained from all subjects except where not required (not required for data analyzed anonymously in Laos PDR at the time of the study). Other clinical isolates were obtained from the China Medical Culture Collection of National Institutes for Food and Drug Control, China, and the National Center for Emerging and Zoonotic Infectious Diseases, Centers for Diseases Control and Prevention, USA. All data were analyzed anonymously.

Culture of leptospires from human blood was performed using EMJH supplemented with 3% rabbit serum and 0.1% agarose, as described previously [9] . Positive cultures were sent to the WHO/FAO/OIE Collaborating Center for Reference & Research on Leptospirosis, Australia for serovar identification using the cross agglutinin absorption test (CAAT) [10] . Definitive identification of species was undertaken by amplification and sequencing of the near full-length 16S rRNA gene. Primers were designed to anneal to conserved regions of genes from pathogenic species L. interrogans, L. kirschneri, L. borgpetersenii, L. santarosai, L. alexanderi and L. fainei. The primers (f - 5′ GTTTGATCCTGGCTCAG 3′ and r -5′CCGCACCTTCCGATAC 3′) amplified a 1,483 bp PCR product which was sequenced in its entirety using internal primer pairs (primers available on request).

WJ460 (DMSO) was purchased from Glixx Laboratories Inc. or MedChem Express; A-485 (DMSO), vacuolin-1 (DMSO) and apilimod (DMSO) were purchased from MedChem Express; EGA (DMSO), leupeptin (H2O) and pepstatin A (DMSO) were purchased from MilliporeSigma; E64D (DMSO) was purchased from Cayman Chemical. Compounds were purchased in solution or reconstituted and were aliquoted and stored at -20°C or -80°C as recommended by the manufacturers. Viability following treatment with WJ460 was assessed using alamarBlue (Bio-Rad) according to the manufacturer’s instructions. Cells were transfected using Turbofect (Thermo Fisher Scientific) according to the manufacturer’s instructions and harvested 24 h later. Whole cell extracts were prepared, and western blotting was done as described [98 (link)]. Affinity enrichment of HBZ using GST-KIX was done as described [34 (link)]. Antibodies used were as follows: anti-HTLV-1 Env (ARP-1578) and anti-Tax (hybridoma 168B17-46-92) were obtained from NIH AIDS Research and Reagent Program; anti-HBZ has been described [99 (link)]; anti-6x His (ab9108) was purchased from Abcam; anti-MyoF (HPA014245) and anti-Myc (06–340) were purchased from MilliporeSigma; anti-HTLV-1 Env gp46 (1C11, sc-53890; 65/6C2.2.34, sc-57865), anti-Gag p19 (TP7, sc-57870) and anti-β-actin (C4, sc-47778) were purchased from Santa-Cruz; anti-clathrin (D3C6, #4796), anti-caveolin-1 (D46G3, #3267), anti-EEA1 (C45B10, #3288), anti-Rab5a (E6N8S, #46449), anti-Rab7 (D95F2, #9367), anti-Rab11 (D4F5, #5589), anti-LAMP-1 (D2D11, #9091) were purchased from Cell Signaling; anti-EHD2 (PA5-80576) was purchased from ThermoFisher Scientific. All blots were developed using Pierce ECL 2 (Thermo Fisher Scientific) and scanned with a Typhoon RGB imager (Cytiva). Immunoblot quantification was performed using ImageQuant TL v8.1 (Cytiva). To enrich Env SU/gp46 when transiently expressed in HEK293T cells, whole cell extracts were immunoprecipitated with Lens Culinary Agglutinin lectin bound to agarose beads (Vector Laboratories) [26 (link)] and beads resuspended in SDS loading dye.

Cells were washed with ice-cold PBS (3×) and lysed in RIPA buffer (contains 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, Cat. No. R0278, Sigma) supplemented with 2 μM TMG, 1 mM PMSF, and 1× protease and phosphatase inhibitors (cOmplete, mini, EDTA-free, and PhosSTOP respectively, Millipore, Sigma). Lysates were sonicated and protein concentration was quantified with the bicinchoninic acid assay (Cat. No. 23225, Pierce). For Western blot, protein samples were combined with 50 mM DTT and 1× LDS sample buffer (NP0008, NuPAGE, Thermo Fisher Scientific), and 10 μg total protein per lane was resolved by electrophoresis, typically run in NuPAGE Bis-Tris 4 to 12% gradient gels (Cat. No. WG1403, Thermo Fisher Scientific). Proteins were transferred onto nitrocellulose membranes (iBlot2 transfer stacks, Cat. No. IB23001, Thermo Fisher Scientific). The membranes were blocked with 5% bovine serum albumin (BSA) in TBS for 1 h at room temperature and then incubated with primary antibodies, typically at a 1000:1 dilution in 5% BSA in 0.1% Tween20-TBS, overnight at 4 °C (see Table S2 for a list of primary antibodies used). For detection, we used the LI-COR Odyssey system and appropriate host-specific secondary antibodies (e.g., IR Dye 800w goat anti-rabbit, Cat. No. 926-32211, LI-COR) typically at a 5000:1 dilution in 5% BSA in 0.1% Tween20-TBS. For the detection of biotinylated targets, we used IRDye800w Streptavidin (Cat. No. 926-32230, 5000:1 in % BSA in 0.1% Tween20-TBS). Quantification of band intensities was performed with Image Studio Lite (LI-COR). Normalization for protein loading was performed based on intensities obtained by staining the membranes with REVERT 700 Total Protein Stain (Cat. No. 926-11010, LI-COR).
For the detection of glycans with lectin blotting, we used lectin kit 1 (Cat. No. BK-1000, Vector Laboratories) that includes biotinylated conjugates of wheat germ agglutinin, ConA, peanut agglutinin, and D. biflorus agglutinin. For the lectiblots, proteins were run on NuPAGE Bis-Tris 4 to 12% gradient gels and then were transferred using the TransBlot Turbo system (Bio-Rad) onto nitrocellulose membranes (kit Cat. No. 1704271, Bio-Rad). Following blocking with 5% BSA in TBS, membranes were incubated with 5000:1 diluted lectins (final concentration of lectin 0.4 μg/ml) overnight at 4 °C. Following extensive washes with TBS-T, the bound biotinylated lectins were detected with IRDye800w Streptavidin. To control for the reactivity of streptavidin with endogenously biotinylated proteins, some experiments were done as negative controls where no lectin was included in the overnight step. These experiments yielded reactive bands at ∼75 and also at 125 and 250 kDa.

To remove nonspecific agglutinins, one volume of 0.5% turkey red blood cells (RBCs) was added to the ferret antisera or human plasma samples, which were then incubated for 1 h at room temperature. After centrifugation at 1200× g rpm for 10 min, the supernatants were collected and examined for nonspecific agglutinins. The supernatants without nonspecific agglutinins were utilized for the following test. The indicated viruses (4 HA units in 25 μL) were incubated with 25 µL of two-fold serially diluted plasma samples at room temperature for 30 min. Then, 0.5% turkey RBCs were added to the mixture and incubated for 30 min at room temperature. Reciprocal numbers of the minimum dilutions of plasma samples required to inhibit hemagglutination were used as the HI titers. Experiments were repeated three times, and representative values were selected based on the three experiments.