Sample collection
Blood and tissue samples were collected during March to September 2017 period from 43 free-ranging small mammals comprising of 39 rats, 1 mouse and 3 shrews inhabiting rural and urban areas in Gampaha district of the Western province of Sri Lanka. Shrews were morphologically identified whilst free ranging rodents were species identified morphologically and also by DNA barcoding techniques. This was followed by PCR screening of orthohantavirus RNA in lung tissues and sera from small mammals captured. Sera of 39 rats were tested additionally for the presence of IgG antibodies to PUUV and SEOV and leptospira genus specific antigens by (ELISA) but sera of three shrews (#14, #35 and #37) and one mouse (#22) were not tested.
Study area was chosen to involve locations from a previous study on hospitalized patients (Figure 1A) with orthohantavirus-like illness and concomitant leptospirosis [29]. The geographic locations where the rodents and shrews were captured are shown in Figure 1B.
Ten live-capture traps were deployed at or above ground level in the afternoon at different locations for trapping small mammals, and the study protocol was approved by the Department of wildlife Conservation in Sri Lanka. Animal handling was carried out following human safety precautions with personal protection equipment (PPE). Live-trapped animals and their locations were recorded by geo-mapping. Euthanasia of live-trapped rodents was conducted after transportation to the laboratory according to The American Veterinary Medical Association (AVMA) guidelines for the euthanasia of animals: 2013 ed. 31 May 2015 [34]. The animals were photographed soon after euthanasia and dissected in a safety hood (category II) for collection of blood (heart puncture) and organs including lung, heart, kidney, liver, spleen, mesenteric lymph nodes, intestines and brain. Tissues and serum samples of 43 animals (40 rodents and 3 shrews) were stored at -80˚C until analysis.
Species identification of captured animals
Chromosomal DNA was extracted from lung tissues of the morphologically identified rodent species. Briefly, individual samples were homogenized with silica beads (1 mm) using MP FastPrep®-24 Instrument (MP Biomedicals, Santa Ana, CA, USA). Samples were thereafter digested with proteinase K (600 mAU/ ml) in buffer G2 (Qiagen Inc, Hilden, Germany) at 56˚C for 15 min, under slow shaking, followed by a quick centrifugation to remove debris. Obtained supernatants were used for DNA extraction and isolation using Qiagen EZ1 Advanced Robotic Workstation (Qiagen Inc). Prior to PCR, primers were designed from available Cytb gene sequences of different local rodent species, see Table 1a.
Table 1: Primers
Primer name
|
Sequence
|
Melting point (Tm)
|
(1a). Cytochrome oxidase b subunit gene (Cytb) primers
|
Cytb 109F
|
CCC ATC CAA CAT CTC ATC ATG ATG A
|
61.3°C
|
Cytb 183F
|
GCC TAT TCC TAG CAA TAC ACT ACA C
|
61.3°C
|
Cytb 515F
|
ACC CTA GTC GAA TGA ATC TGA GG
|
60.6°C
|
Cytb 267R
|
CCG TAG TTT ACG TCT CGG CAG AT
|
62.4°C
|
Cytb 540R
|
CCT CCT CAG ATT CAT TCG ACT AG
|
60.6°C
|
Cytb 669R
|
CCT GTG GGG TTR TTT GAT CCT GT
|
61.5°C
|
(1b) Orthohantavirus primers
|
PanHanta-F1
|
ATG TAT GT [I] AGT GCW GAT GC
|
53.2°C
|
PanHanta-R1
|
ACC A [I] TCW GW [I] CCA TCA YC
|
53.4°C
|
PanHanta-F2
|
TGC WGA TGC [I] ACR AAA TGG TC
|
56.9°C
|
PanHanta-R2
|
GCA TCA TCW GAR TGA TG [I] GCA A
|
57.5°C
|
Seo242F
|
GAC AGG ATT GCA GCA GGG AAG A
|
62.1°C
|
Seo870R
|
CAT CCC TGC AAG TGC ACC TTG
|
61.8°C
|
Seo326F
|
CAC TAA GCT ATG GGA ATA CAC TGG A
|
61.3°C
|
Seo1077R
|
ATG AGG AAC ACA ATC ATG GCT TCA A
|
59.7°C
|
Briefly, nine mitochondrial sequences were downloaded from GenBank and aligned by ClustalW of BioEdit package version 7.1.3.0 [35]. Conserved regions between different rodent sequences were identified and primers within Cytb gene were constructed thereof.
PCR reactions were conducted with different combinations of Cytb primers and extracted DNA samples in standard 96-well plates using KAPA SYBR® FAST qPCR Kit (KAPA Biosystems, Boston, MA, USA) along with CFX96TM 143 Real-time detection system (Bio-Rad Laboratories, Hercules, CA, USA). PCR reaction was carried out in 25 µl mixtures containing 1 µl DNA template, 25 µM of each primer and 12.5µl of KAPA master mix. Initial denaturation at 95°C for 3 min was followed by 40 cycles, each consisting of 5 s at 95°C; 20 s at 55 or 57°C and 15 s at 72°C. Finally, the samples were heated to 95°C for 10 s cooled down to 65°C and re-heated from 65°C to 90°C at a rate of 0.5°C per 5 s.
PCR products were analysed by agarose electrophoresis and purified with illustra MicroSpin S-400 HR Columns TM (GE Healthcare, Sweden) before sequencing the amplicons of samples (Eurofins MWG Operon, Ebersberg, Germany) using amplification primers. Sequence comparisons were finally conducted with corresponding sequences in databases by Standard Nucleotide BLAST analysis.
Detection of orthohantavirus RNA
Total RNA from serum samples or lung tissues were extracted with TRIzol® LS Reagent (InvitrogenTM Life technologies) in a total volume of 1 ml before RNA isolation using RNeasy® Mini Kit and RNeasy® Columns as recommended by the manufacturer (Qiagen, Hilden, Germany). RNA was recovered in 20 µl nuclease-free water. Obtained RNA was stored at −80°C before complementary deoxyribonucleic acid (cDNA) synthesis with GoScript Reverse Transcriptase Kit (Promega, Madison, WI, USA). RT-PCR were carried out using the procedures summarized below; the KAPA SYBR® FAST qPCR Kit (KAPA Biosystems, Boston, MA, USA) and SsoFastTM EvaGreen® Supermix (Bio-Rad, Hercules, CA, USA) kits along with orthohantavirus primers purchased from MWG-Biotech AG (Ebersberg, Germany), see Table 1b.
The basic conditions for PCR kits were those recommended by the manufacturer. Deoxyinosine (dI) were used at sites in the Pan-Hanta primers where more than two different bases are found by a multiple alignment of thirty-three L-segment sequences, representing a wide variety of different orthohantaviruses. Accurate annealing temperatures for primer pairs were investigated individually by temperature gradients starting at 45°C and ending at 55°C. RT-PCR was carried out in 25 µl mixtures containing 1µl cDNA template, 25 µM of each primer and 12.5 µl of KAPA master mix (KAPA SYBR® FAST qPCR Kit, KAPA Biosystems, Boston, MA, USA). An initial denaturation step at 95◦C for 3 min was followed after a total of 35 cycles, each consisting of a denaturation step at 95◦C for 15 s and an annealing temperature of 56◦C for 15 s [36].
Antigen purification
DNA constructs encoding the amino-terminal part of the nucleocapsid (N) protein of PUUV (AY526219) and SEOV (M34881) were expressed from poly-histidine-fusion vectors in Escherichia coli BL-21 DE3 (InvitrogenTM Life technologies). The N proteins were purified using metal chelating chromatography according to a protocol from QIAexpressionist 01/2000 (Qiagen Ltd., UK) [37].
Detection of rat anti- orthohantavirus antibodies
Indirect ELISA was performed on sera collected from 39 rats to determine IgG antibodies to N proteins of PUUV and SEOV. Analysis was essentially done as described earlier [37]. Calculated cut-off value was set to 3 times the optical density (OD) value of negative sera derived from serologically negative laboratory rats (R. norvegicus). Briefly, microtiter plates (Nunc MaxiSorpTM) were coated overnight at 4◦C with 50 µl containing truncated forms of the N proteins (1–3 µg/ ml) in ELISA-coating buffer (carbonate-bicarbonate buffer pH 9.2 to 10.6). Plates were blocked thereafter with 10 mg/ ml of Casein Blocking Buffer (antibodies-online, cat no: ABIN929980) and incubated at 37◦C for 2 hours. Plates were thereafter washed four times with phosphate buffered saline (PBS) containing 0.05% Tween, pipetted 50 µl rat sera diluted 100x in Casein Blocking Buffer supplemented with 0.05% Tween-20® (MERCK, Schuchardt, Germany), incubated for one hour at room temperature. Plates were thereafter washed four times with PBS containing 0.05% Tween, pipetted 50 µl of a secondary horseradish peroxidase (HRP) conjugated goat anti-rat immunoglobulin (H+L) (InvitrogenTM Life technologies), diluted according to the manufacturer’s instructions and incubated 1 h at room temperature. Plates were then washed eight times and finally 100 µl of substrate 3,3’,5,5’- Tetramethylbenzidine (TMB) (Seramun Diagnostica, Heidesee Germany) was added to each well and incubated 15 minutes at room temperature. Reaction was stopped by addition of 0.25 M H2SO4; (100 µl/ well) and OD at 450 nm was determined. Two separate OD measurements with duplicates of each serum were analysed [see Additional file 1].
Detection of rat anti-leptospira IgG
ELISA for quantitative in vitro determination of Rat IgG antibodies to Genus specific leptospira antigens (Creative Diagnostics, USA) was performed on sera from 39 rats and results were interpreted according to manufacturer’s protocol. Sera which gave > 23.5 pg/ ml were considered positive and undetectable levels are below < 23.5 pg/ ml according to manufacturer’s instructions.
Briefly, 50 ml of 1 in 5 diluted rat serum was pipetted to each well of microtiter plate pre-coated with Genus specific leptospira antigens except the blank well and incubated 30 minutes at 37◦C. Thereafter, emptied wells, dried by swing, added 300 μl washing buffer to each well, stilled for 30 s, drained, repeated 5 times and dried by pat. Each well was pipetted 50 μl of ready to use anti-Rat IgG HRP conjugate except the blank well, incubated 30 minutes at 37◦C and washed six times. Then added 50 μl TMB Chromogen Solution A and 50 μl TMB Chromogen Solution B to each well, mixed gently, incubated 15 minutes at 37oC evading light. Added stop solution (50 μl/ well) to each well and read OD at 450 nm within 15 minutes using a Biotek-Ex800 ELISA reader. Concentration of anti-leptospira IgG in rat serum was determined by comparing the OD of the sample with the standard curve derived from ODs to a range of concentrations (150 pg/ ml to 1800 pg/ ml) of the standard reagent (purified rat leptospira IgG) that binds with Genus specific leptospira antigen pre-coated microtitre plate wells in the Rat leptospira IgG ELISA, and the additional figure file shows this in more detail [see Additional file 2]
Research permission and approval to study rodent-borne infectious agents in Sri Lanka was granted by the Department of Wildlife Conservation in Sri Lanka subject to the provisions of Fauna and Flora Protection Ordinance (FFPO) of Sri Lanka (Ref: WL/3/2/85/17) and animal tissues were obtained solely for the purpose of this study.